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Modified atmosphere packaging of pink prawn (Pandalus platyceros) Dheeragool, Panadda 1989

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MODIFIED ATMOSPHERE PACKAGING OF PINK PRAWN (Pandalus platyceros) By PANADDA DHEERAGOOL B.Sc, Kasetsart University, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES Department of Food Science We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA May 1989 © Panadda Dheeragool, 1989 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Panadda Dheeragool Department of Food Science The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date May 1989 DE-6(3/81) i i ABSTRACT Pandalus platyceros ( p i n k p rawn o r s p o t s h r imp ) was s t o r e d unde r (1) a e r o b i c c o n t r o l (bags l e f t open t o a i r ) , (2) c a r b o n d i o x i d e (CMAP), and (3) n i t r o g e n a tmosphe re s (NMAP) a t 1°C. F a c u l t a t i v e anae robes were t he m a j o r i t y o f p s y c h r o t r o p h i c b a c t e r i a w h i c h c o n t a m i n a t e d t he prawns i n t h i s s t u d y . T i s s u e pH o f t h e CMAP prawns was t he l o w e s t o f a l l p rawns and b r o u g h t abou t t h e g r e a t e s t e xuda t i o n o f t he mus c l e t i s s u e . The m i c r o o r g a n i s m s were m a r k e d l y i n h i b i t e d unde r t he CMAP and were s l i g h t l y i n h i b i t e d unde r t h e NMAP. C o n s e q u e n t l y , f o r m a t i o n o f s a l t - s o l u b l e p r o t e i n s , w a t e r - s o l u b l e p r o t e i n s , TMA, and TVB p r o g r e s s e d most s l o w l y i n t he CMAP p r awns . TMA p r o d u c t i o n was f a v o r e d unde r t h e NMAP. No ATP was d e t e c t e d i n t he prawns a t t h e t ime o f p a c k a g i n g . ADP d e g r a d a t i o n was a c c e l e r a t e d unde r t h e CMAP b u t d e l a y e d unde r t he NMAP. AMP deg r aded most r a p i d l y i n t h e c o n t r o l p rawns f o l l o w e d by t he NMAP and t h e CMAP prawns r e s p e c t i v e l y . No e f f e c t o f s t o r a g e a tmosphe res was f ound on IMP, i n o s i n e , and h y p o x a n t h i n e d e c o m p o s i t i o n s . Deve lopment o f K - v a l u e i n prawns was most s t r o n g l y i n f l u e n c e d by IMP d e c o m p o s i t i o n r a t e and was s l o w e r unde r t h e CMAP t h a n unde r t h e NMAP. Redness o f p rawn c o l o u r (Hun t e r a v a l u e ) was m a i n t a i n e d unde r t he NMAP and t h e CMAP. Bu t c a r b o n d i o x i d e i n t he CMAP and t h e NMAP l i k e l y c a u s e d d e s t r u c t i o n o f t h e p i gmen t s d u r i n g s t o r a g e r e s u l t i n g i n l i g h t e r c o l o u r o f t h e s t o r e d prawns (Hun te r L v a l u e ) . S h e l f - l i f e o f t h e p i n k prawns was a t l e a s t d o u b l e d unde r t h e CMAP s y s t em b u t no s h e l f - l i f e e x t e n s i o n was a f f o r d e d by t h e NMAP s y s t e m . The f i r s t p r i n c i p a l component o f the p rawn q u a l i t y p a r ame t e r a c c o u n t e d f o r 59.07% o f t h e d a t a v a r i a n c e . T h i s means t h e most i m p o r t a n t i i i q uality descriptors of the prawns were a l l microbiological variables, raw prawn meat odour and colour scores, TMA, cooked prawn meat odour and colour scores, water-soluble protein and overall sensory score. Stepwise discriminant analysis of pH and t o t a l aerobic sulphide-producing psychrotrophic b a c t e r i a l count data of the prawns with TMAN concentration as the grouping variable, resulted i n 97.2% correct c l a s s i f i c a t i o n of the prawn samples. Two equations for the canonical variables were obtained. A plot of these two canonical variables w i l l v i s u a l l y indicate the region of prawn quality (good, acceptable, or unacceptable). i v TABLE OF CONTENTS Page ABSTRACT i i LIST OF ABBREVIATIONS v i LIST OF TABLES v i i LIST OF FIGURES v i i i ACKNOWLEDGMENTS x i i 1. INTRODUCTION 1 2. LITERATURE REVIEW 6 2.1. Changes During Storage of Prawns Under Normal A i r Environment 7 2.1.1. Change i n Muscle pH 7 2.1.2. Change i n Exudation Formation 8 2.1.3. Changes i n ATP and Its Related Compounds 8 2.1.4. Change i n Total Viable Psychrotrophic Bacterial Count 10 2.1.5. Change i n Total V o l a t i l e Basic Nitrogen Concentration 11 2.1.6. Change i n Trimethylamine-nitrogen Concentration 12 2.1.7. Changes i n Concentrations of Water-soluble and Salt-soluble Muscle Proteins 14 2.1.8. Change i n Colour 15 2.2. Changes Under Carbon Dioxide Modified Atmosphere Storage 18 2.3. Changes Under Nitrogen Modified Atmosphere Storage 22 2.4. Multivariate Analyses 22 2.4.1. Factor Analysis 22 2.4.2. Stepwise Discriminant Analysis 23 3. MATERIALS AND METHODS 25 3.1. Prawn Samples 26 3.2. Packaging Material 26 3.3. Treatments 28 3.4. Sampling Plans 28 3.5. Tests 3.5.1. Headspace Gas Compositions i n the Bags 29 3.5.2. Exudate Determination 30 3.5.3. Microbiological Tests 30 3.5.4. Determination of pHs of Prawn Inner Tissue 35 3.5.5. K-value Determination 35 3.5.5.1. Sample Preparation 35 3.5.5.2. HPLC Conditions 36 3.5.5.3. Reproducibility of the HPLC Analysis 37 3.5.5.4. Internal Standard 37 3.5.5.5. Reproducibility of the Extraction 41 3.5.6. Trimethylamine-nitrogen Determination 41 3.5.7. Total V o l a t i l e Basic Nitrogen Determination 44 3.5.8. Sensory Evaluation 44 3.5.9. Hunter L, a, b Values 57 V 3.5.10. Water-soluble and Salt-soluble Protein Determinations 57 3.6. S t a t i s t i c a l Analyses 57 4. RESULTS 63 4.1. T r i a l 1 4.1.1. Headspace Gas Composition 64 4.1.2. Exudate Formation 64 4.1.3. Tissue pH 70 4.1.4. Microbiology 70 4.1.5. K-values 73 4.1.6. Trimethylamine-nitrogen Concentration 80 4.1.7. Sensory Evaluation 83 4.1.8. Hunter L, a, b Values 92 4.2. T r i a l 2 4.2.1. Headspace Gas Composition 96 4.2.2. Tissue pH 99 4.2.3. Exudate Formation 99 4.2.4. Microbiology 103 4.2.5. Trimethylamine-nitrogen Concentration 108 4.2.6. Total V o l a t i l e Basic Nitrogen Concentration 110 4.2.7. Soluble Proteins 110 4.2.8. Sensory Evaluation 114 4.2.9. Hunter L, a, b Values 123 4.2.10. Factor Analysis 127 4.2.11. Stepwise Discriminant Analysis 131 5. DISCUSSION 134 5.1. Headspace Gas Compositions i n the Bags 135 5.2. Tissue pHs 135 5.3. Exudate Formation 136 5.4. Total Psychrotrophic Bacterial Counts and Total Sulphide-producing Psychrotrophic Bacterial Counts 141 5.5. Trimethylamine-nitrogen Concentration 142 5.6. Total V o l a t i l e Basic Nitrogen Concentration 143 5.7. K-value and Its Related Compounds 144 5.8. Salt-soluble Protein and Water-soluble Protein Concentrations 145 5.9. Colour 146 5.10. Sensory Characteristics 147 5.11. S h e l f - l i f e of the Prawns 148 6. CONCLUSION 150 7. REFERENCES 154 8. APPENDIX A: CHLORAMPHENICOL TREATMENT 163 9. APPENDIX B: SAMPLE MEANS AND STANDARD DEVIATIONS 170 v i LIST OF ABBREVIATIONS a Hunter a value ADP Adenosine diphosphate AMP Adenosine monophosphate ANOVA Analysis of variance ATP Adenosine triphosphate b Hunter b value CC Sensory colour score of cooked prawn meat CF Sensory flavour score of cooked prawn meat CMAP Carbon dioxide modified atmosphere packag(ing/ed) CO Sensory odour score of cooked prawn meat CT Sensory texture score of cooked prawn meat HPLC High performance l i q u i d chromatograph(y/ic) Hx Hypoxanthine HxR Inosine IMP Inosine monophosphate I.S. Internal standard (5-bromouracil) L Hunter L value MAP Modified atmosphere packaging NMAP Nitrogen modified atmosphere packag(ing/ed) OVERALL Mean of a l l sensory scores: Overall = (RC+R0+CC+C0+CF+CT)/6 RC Sensory colour score of raw prawn meat RO Sensory odour score of raw prawn meat TMA Trimethylamine TMAN Trimethylamine-nitrogen TMAO Trimethylamine oxide TSA Total aerobic psychrotrophic b a c t e r i a l count TSN Total anaerobic psychrotrophic b a c t e r i a l count TVBN Total v o l a t i l e basic nitrogen SPA Total aerobic sulphide-producing psychrotrophic b a c t e r i a l count SPN Total anaerobic sulphide-producing psychrotrophic b a c t e r i a l count SSP Salt-soluble proteins WSP Water-soluble proteins v i i LIST OF TABLES Table Page 1 Properties of DUPONT LP 920 packaging f i l m 27 2 Reproducibility of the HPLC column 39 3 K-values of the nucleotide standard solutions with and without the area count of the small unknown peak of the internal standard (n=3) 42 4 Reproducibility of the extraction for K-value determination 43 5 Characteristic changes of pink prawns during spoilage 46 6 F - s t a t i s t i c s from ANOVA on data of T r i a l 1 68 7 F - s t a t i s t i c s from ANOVA on data of individual sampling day of T r i a l 1 69 8 F - s t a t i s t i c s from Friedman two-way ANOVA on sensory data of T r i a l 1 91 9 F - s t a t i s t i c s from ANOVA on data of 12 days storage of T r i a l 2 ... 101 10 F - s t a t i s t i c s from Friedman two-way ANOVA on sensory data of T r i a l 2 122 11 Sorted rotated factor loadings from factor analysis of data of T r i a l 2 128 12 Rotated factor loadings (pattern) from factor analysis of data of T r i a l 2 129 13 Correlation matrix from factor analysis of data of T r i a l 1 137 14 Correlation matrix from factor analysis of data of T r i a l 2 139 v i i i LIST OF FIGURES Figure Page 1 Dimensions of the Hunter L, a, b colour coordinate system 17 2 Chromatogram of the headspace gases i n the CMAP bags 31 3 Chromatogram of the headspace gases i n the NMAP bags 32 4 Anaerobic chamber 34 5 Chromatogram of the nucleotide standard working solution 38 6 Chromatogram of the internal standard 5-bromouracil stock solution 40 7 Sensory evaluation score sheet for cooked prawn meat odour, flavour, and texture scores 48 8 Sensory evaluation score sheet for cooked prawn meat colour score 50 9 Sensory evaluation score sheet for raw prawn meat odour and colour scores 52 10 Temperature gradient at the center of the preheated (200°C) conventional oven 55 11 Temperature gradient inside the edible prawn tissue during cooking at 200°C 56 12 Multivariate data input format 59 13 T r i a l 1: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the CMAP bags stored at 1°C 65 14 T r i a l 1: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the NMAP bags stored at 1°C 66 15 T r i a l 1: Exudate volumes of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 67 16 T r i a l 1: Inner tissue pHs of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at TC 71 17 T r i a l 1: Total aerobic and t o t a l anaerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 72 18 T r i a l 1: K-values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 74 i x 19 T r i a l 1: Adenosine diphosphate concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 75 20 T r i a l 1: Adenosine monophosphate concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 77 21 T r i a l 1: Inosine monophosphate concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 78 22 T r i a l 1: Inosine concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 79 23 T r i a l 1: Hypoxanthine concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 81 24 T r i a l 1: Trimethylamine-nitrogen concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C ' 82 25 T r i a l 1: Overall sensory scores of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at TC 84 26 T r i a l 1: Scores for raw prawn meat odour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 85 27 T r i a l 1: Scores for cooked prawn meat odour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 86 28 T r i a l 1: Scores for cooked prawn meat colour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 87 29 T r i a l 1: Scores for cooked prawn meat texture of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 88 30 T r i a l 1: Scores for raw prawn meat colour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 89 31 T r i a l 1: Scores for cooked prawn meat flavour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 90 32 T r i a l 1: Hunter L values of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 93 33 T r i a l 1: Hunter a values of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 94 X 34 T r i a l 1: Hunter b values of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 95 35 T r i a l 2: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the CMAP bags stored at 1°C 97 36 T r i a l 2: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the NMAP bags stored at 1°C 98 37 T r i a l 2: Inner tissue pHs of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 100 38 T r i a l 2: Exudate volumes of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 102 39 T r i a l 2: Total aerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 104 40 T r i a l 2: Total anaerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 105 41 T r i a l 2: Total aerobic sulphide-producing psychrotrophic b a c t e r i a l counts of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 106 42 T r i a l 2: Total anaerobic sulphide-producing psychrotrophic b a c t e r i a l counts of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 107 43 T r i a l 2: Trimethylamine-nitrogen concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 109 44 T r i a l 2: Total v o l a t i l e basic nitrogen concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C I l l 45 T r i a l 2: Water-soluble protein concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 112 46 T r i a l 2: Salt-soluble protein concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 113 47 T r i a l 2: Overall sensory scores of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 115 48 T r i a l 2: Scores for raw prawn meat colour of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 116 x i 49 T r i a l 2: Scores for raw prawn meat odour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 117 50 T r i a l 2: Scores for cooked prawn meat colour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 118 51 T r i a l 2: Scores for cooked prawn meat odour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 119 52 T r i a l 2: Scores for cooked prawn meat flavour of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 120 53 T r i a l 2: Scores for cooked prawn meat texture of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 121 54 T r i a l 2: Hunter L values of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 124 55 T r i a l 2: Hunter a values of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 125 56 T r i a l 2: Hunter b values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 126 57 T r i a l 2: Plots of Factor 1 against Factor 2 of rotated factor loadings from the factor analysis on data of 12 days storage .... 130 58 T r a i l 2: Canonical plots of prawn samples c l a s s i f i e d prawns by stepwise discriminant analysis of pH and SPA 133 x i i ACKNOWLEDGEMENT I wish to express my sincere gratitude to my thesis supervisor Dr. B.J. Skura for a l l h is time, patience, guidance, and understanding. I also wish to thank the members of my graduate committee: Drs. W.D. Powrie, P. Townsley, S. Nakai, and G. Strasdine, for a l l their valuable suggestions and guidance. For technical assistance and advice, I am greatful to Dr. R. Wu, Sherman Yee, and Valerie Skura. For advice i n s t a t i s t i c a l analyses, I thank Dr. N. Heckman, Li s a Kan, Calvin L a i , and Stanley K i t a . I thank Dr. Eunice Li-Chan for assistance i n using the BMDP program. I am greatful to Seema Datta and Sunny Lee for their help i n packaging 150 lbs of prawns and for proofreading this thesis. I thank my sensory panel for their enthusiastic p a r t i c i p a t i o n . My appreciation also goes to the departmental secretaries Joyce Tom and Charlene Campbell. Very special thanks goes to Murray W i l l i n g for his endless encouragement and supports, and for making my stay i n Canada the most wonderful thing i n my l i f e . My gratitude also goes to his loving parents and their family members who always make me fe e l so much at home. I thank both the Canadian and Thai Governments for arranging this scholarship program under the ASEAN-CANADA Fisheries Post-Harvest Technology Project. INTRODUCTION 2 1. INTRODUCTION Quality deterioration of a fresh fishery product results from a number of different factors. Bacterial action releases metabolites causing off-odours which are associated with spoilage. Therefore, control of b a c t e r i a l a c t i v i t y i s the key to extend the s h e l f - l i f e of the product. Applications of modified atmosphere packaging (MAP) systems on fishery products were f i r s t published by Coyne (1932 and 1933). Carbon dioxide storage atmosphere was found to extend s h e l f - l i f e of the f i s h at 15°C and s h e l f - l i f e was further extended when the storage temperature was at 2°C. A modified atmosphere packaging (MAP) system i s an enclosed system i n which the composition of the gaseous environment i n the package d i f f e r s from the composition of normal a i r . The gas composition i s normally predetermined at the time of packaging. Once the package i s sealed, there i s no external control of the atmosphere. The p l a s t i c films used for modified atmosphere packages (MAP) usually have gas barrier properties to help maintain atmospheric composition within the packages i n a desired range. Most of the storage atmospheres applied to fishery products were primarily enriched with carbon dioxide gas, as a means of con t r o l l i n g b a c t e r i a l growth, resulting i n the preservation of the products. Commercially, MAP system for fishery products employs a gas mixture of carbon dioxide and nitrogen rather than carbon dioxide alone since carbon dioxide dissolves i n the aqueous phase of the product tissue and causes package collapse (Lannelongue et al., 1982). Further, 3 carbon dioxide causes a s i g n i f i c a n t weight loss of the product due to the formation of large amounts of exudate. Besides the lengthening the s h e l f - l i f e of the fishery product, the MAP system has other advantages such as: improved quality maintenance, extended t r a n s i t time, less problems with melting i c e , better access to the r e t a i l market, active i n h i b i t i o n of b a c t e r i a l and fungal growth, and reduced economic losses (Wolfe, 1980; Smith, 1987). Disadvantages include: added cost; variable product requirements; equipment and personnel training requirements; need for good quality raw material, maintenance of package i n t e g r i t y , and good temperature control (Wolfe, 1980; Smith, 1987). Storage temperatures below 2°C (Smith, 1987) must be constantly maintained to prevent the growth of non-proteolytic Clostridium botulinum whose toxin can cause botulism. This toxin i s heat sensitive and i s destroyed when the product i s cooked properly. Pink prawn, Pandalus platyceros, i s the largest of the l o c a l prawns harvested i n B r i t i s h Columbia. I t now ranks f i r s t i n landed value i n the prawn fishery (Department of Fisheries and Oceans, Canada/1359 UW/29E). The common name for Pandalus platyceros, recognized by the F.A.O. and the U.S.A., i s spot shrimp. I t i s also larger than most of the P a c i f i c pan-da l i d shrimp. The species inhabits the Northern P a c i f i c Ocean from the Bering S t r a i t s to Japan and Korea i n the west, to Southern C a l i f o r n i a i n the east (Dore and Frimodt, 1987). Its body color i s usually reddish brown or tan with white horizontal bars on the carapace, and has the di s t i n c t i v e white spots on the f i r s t and f i f t h abdominal segments (Department of Fisheries and Oceans/1359 UW/29E). Previous studies on headless, shell-on penaeid shrimps stored at refr i g e r a t i o n temperature, under a i r environment (Cobb et a l . , 1973; and 4 Lannelongue et a.1., 1982) showed that the s h e l f - l i f e of shrimp from the Gulf of Mexico was only about a week or l e s s . By using a carbon dioxide enriched atmosphere as the storage atmosphere at r e f r i g e r a t i o n temperature of 4°C, the s h e l f - l i f e of the shrimp was extended (Lannelongue et al., 1982). One hundred percent carbon dioxide as the storage atmosphere doubled the s h e l f - l i f e of these shrimps to 14 days. This extended s h e l f - l i f e i s b e n e f i c i a l . Since the prawns are available only 6 months of the year, the demand for this prawn species i s great, and the price i s high, extended s h e l f - l i f e means a longer marketing period. The extended s h e l f - l i f e also makes transportation of the prawns to remote areas i n fresh form possible. Up to the present time, more information on the effects of carbon dioxide and nitrogen as storage atmospheres on the quality of the prawns i s s t i l l needed. In the marketplace, i t i s essential to have some freshness or spoilage indicator(s) that can be r e l i a b l y used to help establish the quality of the prawns as well as other fishery products that were stored under a MAP system. I t i s also very important to be able to use these spoilage indicators as a means to predict spoilage as well as s h e l f - l i f e l i m i t of the prawns. Several investigations on MAP storage of fishery products used t o t a l b a c t e r i a l count, TVBN, and TMAN concentrations to evaluate changes i n many fishery products including shrimp (Lannelongue et al., 1982; Parkin et al., 1982; Layrisse amd Matches, 1984). However, other indicators, such as K-value, Hunter L, a, b values, and sulphide-producing psychrotrophic bacteria have not yet been explored. 5 The objectives of this research were: (1) to measure the effects of carbon dioxide modified atmosphere packaging (CMAP) and nitrogen modified atmosphere packaging (NMAP) systems on the microbial, chemical, and sensory changes in the pink prawn; and (2) to statistically determine an indicator or a combination of in-dicators that can be effectively used for predicting the spoilage or shelf-life of pink prawns. LITERATURE REVIEW 7 2. LITERATURE REVIEW 2.1 Changes during storage of prawns under normal air environment. The process of f i s h spoilage has been found to be rather complicated as i t i s caused by a number of interrelated systems. In general, after a f i s h dies, i t s respiration and thus i t s oxygen supply stops. Spoilage passes through the stages of rigor mortis, dissolution of rigor mortis, and autolysis respectively. Glycolysis proceeds with the breakdown of glycogen by tissue enzymes followed by the formation of l a c t i c acid through a series of reactions. The accumulation of l a c t i c acid causes a pH drop i n the muscle and induces the l i b e r a t i o n and a c t i v a t i o n of inherent acid proteases. Proteolytic a c t i v i t i e s of tissue enzymes and microbial enzymes result i n the formation of non-protein nitrogenous compounds and amino acids. ATP degradation proceeds during the death struggle and immmediately after death, eventually r e s u l t i n g i n hypoxanthine accumulation. These autolytic products support the growth of microorganisms that contaminate the animal. A number of v o l a t i l e , low molecular weight compounds are formed as a result of microbial actions and contributed to spoilage off-odour. During storage under normal a i r environment, changes that occur af-ter the death of the f i s h that have been used to indicate quality of the edible tissues are reviewed below. 2.1.1 . Change in muscle pH Glycolysis i s the process i n which glycogen i s broken-down to l a c t i c acid and as a consequence, tissue pH i s lowered. Even though the pattern of decrease of glycogen and increase of l a c t i c acid i n shrimp are similar 8 to those of other marine and land animals ( F l i c k and L o v e l l , 1972), i n many studies on shrimp, the absence of an i n i t i a l postmortem drop i n pH has also been reported (Layrisse and Matches, 1984; F l i c k and L o v e l l , 1972; Bailey et al. , 1956). This may be one unique feature of shrimp muscle. I t may be because the glycogen levels i n shrimp are lower than those i n f i s h ; or i t may be associated with a release of low molecular weight bases i n the tissue soon after death, as crustacean muscle contains higher concentrations of non-protein nitrogenous compounds than f i s h and mammals ( F l i c k and L o v e l l , 1972). 2.1.2. Change in exudation formation This decreasing pH causes some of the sarcoplasmic proteins to aggregate and form a pr e c i p i t a t e . The pr e c i p i t a t i o n of these proteins onto m y o f i b r i l l a r proteins causes the s o l u b i l i t y and water-holding capacity of m y o f i b r i l l a r proteins to decrease (Khan, 1977). As a result of the decreasing water-holding capacity of the myofi-b r i l l a r proteins, a portion of water separates from the muscle c e l l s . This portion of water i s cal l e d exudate or drip. In porcine muscle, a pH lower than 6.1 appears to be the c r i t i c a l point where the exudation increases (Warriss, 1982; Warriss and Brown, 1987). However, no information on c r i t i c a l pH of prawn muscle has been found i n the l i t e r a t u r e . 2.1.3. Change in ATP and related compounds During the death struggle and after death, ATP i n f i s h muscle breaks down through the following autocatabolic pathway: ATP -» ADP -»• AMP -> IMP -»• HxR -+ Hx 9 Ehira (1976) demonstrated that inosine and hypoxanthine formation rates d i f f e r among f i s h species due to the a c t i v i t i e s of the corresponding enzymes: nucleoside hydrolase and nucleoside phosphorylase, respectively. For the same reason, Saito et a l . , (1959) suggested the use of the inosine and hypoxanthine portion, as a r a t i o of the t o t a l nucleotide f r a c t i o n , to measure f i s h freshness. The r a t i o was expressed as % K-value which can be calculated from the following equation: HxR + Hx~ % K-value = x 100 ATP + ADP + AMP + IMP + HxR + Hx Among the species of f i s h tested by Ehira (1976), K-values coincided with freshness of the f i s h . He also mentioned that the K-value was influenced most strongly by the decomposition rate of IMP which was related to the a c t i v i t y of phosphatase i n f i s h muscle. Two recent studies on the degradation of ATP showed that IMP dephosphorylation was not dependent on ba c t e r i a l actions while Hx forma-tion was affected by ba c t e r i a l enzymes. Fletcher et a l . , (1988) studied orange roughy stored at 5°C with and without inoculation with Pseudomonas fragi. Surette et al., (1988) evaluated s t e r i l e and non-sterile cod f i l l e t s placed on ice and held at 2-5°C. Fletcher et a l . , (1988) found that while breakdown of purine derivatives to inosine was not affected by delayed f i l l e t i n g , breakdown of inosine to hypoxanthine as well as bac t e r i a l spoilage were delayed with delayed f i l l e t i n g . Surette et a l . , (1988) found that both Pseudomonas spp. and Proteus spp. were responsible for the production of i n t r a c e l l u l a r inosine nucleosidase and hypoxanthine production was more pronounced i n f i l l e t s than i n gutted whole f i s h . Kosak and Toledo (1981) found that, by using chlorine pretreatment, 10 hypoxanthine accumulation i n eviscerated, headless, scaled mullet was delayed. Therefore, as a conclusion, K-value increased gradually by auto-degradation and then increased rapidly as a result of ba c t e r i a l a c t i v i t y (Kosak and Toledo, 1981; Fletcher et a l . , 1988; Surette et a l . , 1988). Kasemsarn et a l . , (1962) found IMP to be a flavor enhancer and hypoxanthine to impart a b i t t e r taste. As i n other marine species, the i n i t i a l l e v e l of IMP i n shrimp muscle, was considerably higher than that found i n mammalian muscle even though the i n i t i a l l e v e l of ATP i n shrimp muscle was similar to that of marine and land vertebrates ( F l i c k and Lo v e l l , 1972). 2.1.4. Total viable bacterial count The majority of natural f l o r a i n f i s h and crustaceans are gram-nega-t i v e , non-fermentative bacteria (Nickelson and Finne, 1984). The number and types of these organisms depend on area of catch, season, and harvest method. The type of spoilage f l o r a w i l l depend on the type of microorganisms that contaminate the f i s h and how the f i s h was handled. Pseudomonas spp., Alteromonas putrefaciens, and Enterobacteriaceae are common natural and spoilage f l o r a i n f i s h and crustaceans. When these organisms reached certain populations, their proteolytic a c t i v i t i e s were s u f f i c i e n t to invade underlying muscle tissue of the animal. At the late logarithmic phase of microbial growth amino acids were released due to the proteolysis of muscle proteins by these b a c t e r i a l enzymes and then u t i l i z e d . Microbial catabolism resulted i n formation of hydrogen sulphide, organic sulphide, amines, and ammonia which have rather offensive smells. I t was when this microbial proteolysis took place that 11 spoilage began. Therefore, the t o t a l population of these l i v i n g bacteria, also c a l l e d t o t a l viable b a c t e r i a l count, has been used to indicate spoilage onset i n f i s h . 2.1.5. Change in total volatile basic nitrogen concentration An increase i n TVBN l e v e l i n shrimp muscle during postmortem storage on ice has been reported by several investigators. V o l a t i l e bases can be produced by both natural shrimp tissue enzymes and ba c t e r i a l enzymes (Cobb and Vanderzant, 1971; Yeh et al., 1978). Amino acids i n shrimp muscle are converted to the corresponding amines by decarboxylases. Later, ammonia i s liberated by amine oxidase. Ammonia was found to be the primary component i n the TVB fracti o n (Vanderzant et a l . , 1973). Production of ammonia by tissue enzymes was dependent on pH and temperature (Yeh et a l . , 1978). Two optimal pHs of 6.0 and 8.5 and an optimal temperature of 37°C were reported. Bacterial decarboxylases were active under acidic conditions while amine oxidase was able to act over a wide pH range from s l i g h t l y acidic to alkaline (Satake et a l . , 1952, cit e d i n Tomiyasu and Zenitani, 1957, no sp e c i f i c pH range revealed). Among a number of ammonia-producing enzymes tested by Yeh et a l . , (1978), only adenosine deaminase and AMP deaminase were found to be present at si g n i f i c a n t levels i n white shrimp muscle. White shrimp, inoculated with Pseudomonas species and stored for 11-14 days at 5°C, had higher levels of TVBN than the corresponding con-t r o l (Cobb and Vanderzant, 1971). Gagnon and Felle r s (1958) found a good correlation between TVBN and t o t a l b a c t e r i a l count, and between TVBN and the sensory panel scores of shrimp. I t was often reported that TVBN increased s i g n i f i c a n t l y only after the product had already spoiled 12 (Jacober and Rand, 1982). Therefore, i t did not r e f l e c t a l l the quality stages of the prawns but rather was indicative of advanced deterioration and spoilage. 2.1.6. Change in trimethylamine nitrogen concentration Generally, TMA i s detected only i n marine f i s h and not i n freshwater f i s h (Harada, 1975). The presence of TMA i n freshly caught f i s h p r i o r to the onset of the ba c t e r i a l growth has been reported. However, i n most cases i t was found i n extremely low concentrations, normally under 1 mg TMAN/lOOgm (Hebard et a l . , 1982). Trimethylamine oxide (TMAO) i s the precursor of TMA. TMAO i s re-duced to TMA and to dimethylamine and formaldehyde mainly by exogenous ba c t e r i a l enzymes during spoilage of f i s h including shrimp. Bacteria capable of reducing TMAO include most species of Enterobacteriaceae. Among these are Pseudomonas, the natural f l o r a of f i s h and s h e l l f i s h . Pseudomonas species are known to be mainly responsible for spoilage of fresh f i s h at low temperatures which favor their growth. A linea r relationship between Alteromonas putrefaciens population and TMA production was demonstrated by Laycock and Regier (1971). Therefore, the usefulness of TMA production as an indicator of f i s h quality depends on the presence of TMA-producing organisms such as, Alteromonas putrefaciens, i n the spoilage f l o r a . The optimum temperature for TMAO reduction by each microorganism i s different (Sasajima, 1973). TMA production occurs very slowly at c h i l l temperatures but proceeds more rapidly at room temperature. There i s a lag period i n TMA formation i n f i s h stored at low temperatures, and the lower the temperature, the longer the lag period (Anderson and F e l l e r s , 13 1949). At subzero temperatures, where the bacteria f a i l to reach the necessary c e l l counts to start TMAO reduction, TMA production i s almost t o t a l l y i n h i b i t e d (Sasajima, 1973, 1974). TMA upon reaction with l i p i d i n the f i s h muscle, produces the fishy odour (Davies and G i l l , 1936). The experiment on cod muscle press juice by Beatty and Gibbons (1937) revealed that TMA concentration, which was almost negligible i n fresh f i s h , increased readily at the onset of the spoilage and correlated with odours. In contrast, no correlation between TMA concentrations and sensory scores (flavour and odour) was found i n roundnose grenadier (white fish) (Botta and Shaw, 1976). TMA i s usually formed under anaerobic conditions as a result of anaerobic respiration of bacteria which u t i l i z e s TMAO (Watson, 1939). This was probably because a ba c t e r i a l enzyme trimethylamine oxide reduc-tase, which reduces TMAO to TMA, was active only under anaerobic condi-tions as reported by Easter et al. (1982). Greater TMA production was also evident when a low oxygen permeable packaging material was used (Murray et al., 1971). I t i s obvious that a f i l m with very low oxygen permeability favors anaerobic growth and prevents aerobic growth. Thus the use of this type of packaging f i l m may promote this type of spoilage to the maximum. It had also often been reported that TMA appeared i n si g n i f i c a n t amounts only i n the late stages of the seafood quality period (Jacober and Rand, 1982) . Thus i t was rather indicative of advanced deterioration and spoilage and did not r e f l e c t a l l the quality stages during storage of seafood products. 14 2.1.7. Changes in concentrations of water-soluble and salt-soluble  muscle proteins Another method for evaluating the extent of spoilage i s to measure the concentrations of water-soluble and salt-soluble proteins i n muscle. According to Goll et al., (1977), proteins i n muscle c e l l s can be c l a s s i f i e d into three groups based on the i r s o l u b i l i t i e s : sarcoplasmic (proteins i n muscle plasma), stroma (connective t i s s u e ) , and m y o f i b r i l l a r (contractive tissue) proteins. The sarcoplasmic proteins are water-extractable. M y o f i b r i l l a r proteins are actin and myosin which form the myof i b r i l s . The m y o f i b r i l l a r proteins can be extracted with high ionic strength solutions. The stroma proteins comprise the connective tissue (collagen and elastin) and cannot be extracted with water, acid, a l k a l i , or neutral s a l t solutions. According to S p i n e l l i and Dassow (1982), f i s h muscle contains approximately 20-30% of sarcoplasmic proteins, 70-80% of m y o f i b r i l l a r proteins, and a negligible amount of stroma proteins. Muscle protein s o l u b i l i t i e s change with time after an animal's death. These changes are due to the effect of storage temperature on muscle proteinases and other changes occurring i n the f i s h muscle, such as pH (Greaser, 1986). Changes i n protein s o l u b i l i t i e s r e f l e c t : i r r e v e r s i b l e denaturation due to postmortem pH-temperature combinations; proteolysis; reversible p r e c i p i t a t i o n due to lower pH; or altered protein-protein interactions as a result of changes i n the concentrations of small molecular weight substrates (for example, interaction of myosin and a c t i n with ATP). Bacteria, which possess a very high proteolytic a c t i v i t y , can cause extensive breakdown of these muscle proteins (Hasegawa et al., 1970). Pseudomonas tragi grew most rapidly compared to the other meat spoilage 15 b a c t e r i a (Pediococcus cerevisiae, Leuconostoc mesenteroides, and s a l t -t o l e r a n t Micrococcus luteus) on p o r c i n e mus c l e s t o r e d a t 2°C and 10°C ( B o r t o n e t al., 1 9 7 0 ) . Pseudomonas tragi i n c r e a s e d t h e w a t e r - s o l u b l e p r o t e i n s l e v e l i n p o r c i n e musc l e a t t h e l a t e r p e r i o d o f s t o r a g e w h i l e t h e o t h e r m i c r o o r g a n i s m s d i d n o t . The p o r c i n e mus c l e i n o c u l a t e d w i t h Pseudomonas tragi h ad g r e a t e r amounts o f s a l t - s o l u b l e and w a t e r - s o l u b l e p r o t e i n s compared t o t h e c o r r e s p o n d i n g c o n t r o l s . S i m i l a r f i n d i n g s f o r b e e f mus c l e were r e p o r t e d by Yada and S k u r a ( 1 9 8 1 ) . Wh i t e s h r i m p , i n o c u l a t e d w i t h f l u o r e s c e n t Pseudomonas, was t h e o n l y s amp l e , among o t h e r samp les i n o c u l a t e d w i t h d i f f e r e n t m i c r o o r g a n i s m s (Bacillus and c o r y n e f o r m b a c t e r i a ) , t h a t showed a ma jo r i n c r e a s e i n w a t e r - s o l u b l e p r o t e i n s (Cobb and V a n d e r z a n t , 1 9 7 1 ) . A t t h e end o f t h e i r e x p e r i m e n t , t h e r e was l e s s s a l t - s o l u b l e p r o t e i n i n t he sh r imp t i s s u e i n o c u l a t e d w i t h t h e f l u o r e s c e n t Pseudomonas t h a n i n t h e c o r r e s p o n d i n g c o n t r o l . Compared t o o t h e r meat s p o i l a g e b a c t e r i a , pseudomonads grew most r a p i d l y and t h e i r p r onounced p r o t e o l y t i c a c t i v i t y c a u s e d e x t e n s i v e b reakdown o f mus c l e p r o t e i n s , r e s u l t i n g i n h i g h c o n c e n t r a t i o n o f s a l t - s o l u b l e p r o t e i n s a nd , l a t e r on , w a t e r - s o l u b l e p r o t e i n s . 2.1.8. Change in colour C o l o u r i s one o f t h e ma jo r q u a l i t y a t t r i b u t e s i n f o o d . Consumers e x p e c t a p a r t i c u l a r f o o d t o have a c e r t a i n c o l o u r and t h e y a l s o a s s o c i a t e t he s p e c i f i c c o l o u r w i t h t he q u a l i t y o f t he f o o d . S i n c e i t i s e a s i e r t o j u dge c o l o u r t h an f l a v o u r o r t e x t u r e , c o l o u r i s o f t e n u s e d as a measurement o f f o o d q u a l i t y . However , d e s c r i b i n g a c o l o u r i s n o t e a s y . A more m e a n i n g f u l d e s c r i p t i o n o f c o l o u r c a n be a c h i e v e d by e x p r e s s i n g t he 16 colour i n 3 scales: lightness to darkness; hue, such as green, yellow, and red; and intensity of colour. To examine the relationship between the colour of raw and canned sockeye salmon f l e s h , Schmidt and Idler (1958) successfully used a colour scale c a l l e d a value which ranges from redness to greenness, From the r e s u l t s , they were able to predict the colour of canned sockeye salmon from the colour of the raw f l e s h . Three suggested colour grades of sockeye salmon were based on the a value. Hunter colour measurement was developed as a means to provide reasonably uniform estimates of perceived colour intervals or colour relationships adequately representing the colour of objects. Hunter co-lour scales describe colour of an object i n 3 terms: L i s a scale of whiteness (100) to blackness (0); a i s a scale of redness (100) to green-ness (-80); and b i s a scale of yellowness (70) to blueness (-80). The dimensions of the Hunter L, a, b, colour coordinate system are i l l u s t r a t e d i n Figure 1 (Woyewoda et al., 1986). Red colour of prawns i s mainly due to astaxanthin, the major carotenoid pigment i n prawns (sh e l l and f l e s h ) . Prawns discolour when the carotenoid pigments are degraded. Oxidation or hydrolysis of these pigments can be caused by l i g h t , oxygen, heat, and pH conditions. Carotenoid pigments can also be converted by lipoxygenase to colourless end products. 17 Figure 1. Dimensions of the Hunter L, a, b colour coordinate system 18 2.2. Changes under carbon dioxide modified atmosphere storage Under a carbon dioxide packaging atmosphere, a decline i n the pH of a meat product occurred during the i n i t i a l storage period (Parkin and Brown, 1982). The higher the carbon dioxide concentration i n the input gas, the greater the pH drop of meat. The pH drop occurred at the same time as the carbon dioxide concentration i n the headspace gas i n the package decreased (Lannelongue et a l . , 1982). The progression of carbon dioxide absorption into muscle tissue was demonstrated by Wang and Ogridziak (1986). I t was speculated that carbonic acid (the s o l u b i l i z e d carbon dioxide) i n the l i q u i d phase of the treated tissue may have some negative effects on various enzymatic and biochemical pathways required for growth and metabolism i n microorganisms or i n the muscle tissue (Da-ni e l s et a l . , 1985). The combined effect of these metabolic i n t e r f e r -ences may constitute a stress on the system, resulting i n a slowing of bac t e r i a l growth rate. Daniels et a l . , (1986) demonstrated that carbonic acid extended s h e l f - l i f e of cod f i l l e t s at 2°C. Haines (1933) reported that carbon dioxide increased the microbial lag phase and slowed growth rate of certain microorganisms during their logarithmic phase. Effects of carbon dioxide modified atmosphere storage of fishery products increased as storage temperature decreased because as temperature decreases, the s o l u b i l i t y of carbon dioxide i n an aqueous system increases. This i s the reason that MAP s t i l l requires r e f r i g e r a t i o n regulation. Nevertheless, i n combination with r e f r i g e r a t i o n temperature, the inhibito r y effect of carbon dioxide on meat spoilage bacteria varied among bac t e r i a l species ( G i l l and Tan, 1980). Pseudomonas species grew very rapidly under an a i r environment, but were t o t a l l y i n h i b i t e d or k i l l e d under a carbon dioxide atmosphere 19 (Wang and Ogridziak, 1986). In contrast, Lactobacillus spp. and tan Alteromonas spp. grew slower i n modified atmospheres than i n a i r , but outgrew Pseudomonas spp. i n modified atmosphere conditions. As a res u l t of the retarded growth of Pseudomonas spp. under carbon dioxide environment, spoilage was delayed. While aerobic spoilage microorganisms are inhibited by carbon diox-ide at concentrations as low as 20% (Haines, 1933), carbon dioxide does not appear to have a s i g n i f i c a n t i n h i b i t o r y effect on the growth of anaerobic bacteria (Huffman, 1974). I t i s also u n l i k e l y that facultative anaerobic food pathogens, such as Staphylococcus aureus and Salmonella, which grow very slowly, i f at a l l , at re f r i g e r a t i o n temperature, w i l l grow under a carbon dioxide enriched atmosphere even at an abusive temperature such as 10°C (Gray et a l . , 1983). Lactic acid bacteria are anaerobic and aerotolerant ( G i l l , 1986). They u t i l i z e amino acids such as valine and leucine resulting i n the formation of v o l a t i l e f a t t y acids which give cheesy odour. Spoilage caused by l a c t i c acid bacteria, therefore, was not noticeable u n t i l long after the t o t a l viable b a c t e r i a l count had maximized. Layrisse and Matches (1984) studied changes of Pandalus platyceros stored under 50% carbon dioxide (balance a i r ) and 100% carbon dioxide. They compared these changes to the changes i n prawns stored under 100% a i r . From thei r report, on the day of catch, the microbial f l o r a was composed of a variety of bacteria. Sixty-eight percent of the microbial population were gram positive bacteria: Lactobacillus-like organisms and coryneforms. The rest were gram negative bacteria: Flavobacterium, Pseudomonas, and Enterobacteriaciae. 20 During storage under these MAP systems, gram positive bacteria be-came predominant. I t was found that 100% carbon dioxide MAP system i n -creased the microbial lag phase to 8 days. Layrisse and Matches (1984) concluded that, based on b a c t e r i a l counts (between log1 0 6.0 to 6.5) and ammonia (approximately up to 50 mgX) concentration, s h e l f - l i f e of the headless, shell-on prawns was up to 16 days for the MAP systems and about 14 days for the control. The control samples i n the study by Layrisse and Matches (1984) had a longer s h e l f - l i f e than expected because the bags (made of heat-sealable polyester/polyolefin with moisture vapour transmission rate, oxygen transmission rate, and carbon dioxide transmission rate of 0.1 gm, 1 cc, and 27 cc/lOOin /24hr respectively) were sealed. Microbial respiration inside the bags resulted i n the build-up of carbon dioxide. Thus, the atmosphere inside the control sample bags gradually became enriched with carbon dioxide and eventually became a carbon dioxide modified atmosphere system. Nevertheless, i n another experiment by Matches and Layrisse (1985), s h e l f - l i f e of Pandalus platyceros stored at 1-2°C i n ice under normal a i r environment was s t i l l about 2 weeks based on sensory c r i t e r i a . The b e n e f i c i a l effects of carbon dioxide i n con t r o l l i n g microbial growth result i n the extended storage l i f e of shrimp (Barnett et a l . , 1978; Bullard and C o l l i n s , 1978; Lannelongue et a l . , 1982; Layrisse and Matches, 1984; Matches and Layrisse, 1985). The in h i b i t o r y effect of carbon dioxide on microbial growth was due to the acidic condition i n -duced by the dissolved carbon dioxide i n the aqueous phase of the muscle tissue (Wang and Ogrydziak, 1986). 21 Bullard and Co l l i n s (1978) reported that peeled pink shrimp Pandalus borealis stored i n carbon dioxide modified refrigerated seawater at -1.7°C developed lower concentrations of t o t a l v o l a t i l e bases and trimethylamine than those held i n i c e . Moreover, the colour of shrimp held i n carbon dioxide modified refrigerated seawater was much better than that of shrimp held i n i c e , as indicated by a higher carotenoid index i n the shrimp stored i n carbon dioxide modified refrigerated seawater (Nelson and Barnett, 1971; Bullard and C o l l i n s , 1978). Carbon dioxide modified atmosphere (80% carbon dioxide, remaining a i r ) also e f f e c t i v e l y reduced formation of amines (histamine, tyramine, cadavarine, and putrescine) i n whole P a c i f i c mackerel stored at abusive temperature of 20°C to about ha l f of that found i n mackerel stored i n a i r (Watts and Brown, 1982). Wang and Brown (1983) found that concentrations of ammonia, trimethylamine, and t o t a l plate counts were lower i n crayfish stored under 80% carbon dioxide (balance a i r ) compared to samples stored under a normal a i r environment, after 28 days of storage. No differences i n surface colour and odour between fresh rock cod f i l l e t samples and samples stored 13 days i n the modified atmosphere were detected by trained sensory panelists, while both groups were s i g n i f i c a n t l y different from the a i r control (Parkin and Brown, 1982). I t i s obvious that p l a s t i c f i l m with low d i f f u s i o n c o e f f i c i e n t s for gases w i l l be more appropriate to maintain a given atmosphere composition throughout the storage period. Packaging films also affect the storage l i f e of the f i s h products. As demonstrated by Debevere and Voets (1971), the greater the oxygen permeability of the f i l m , the longer the shelf-l i f e of the f i s h . In agreement with Murray et a l . , (1971), the larger supply of oxygen favored aerobic degradation but i t did not favor TMA 22 production. Nevertheless, the rate of deterioration considered from bacteriological and chemical q u a l i t i e s ( t o t a l aerobic b a c t e r i a l counts, t o t a l TMAO-reducing b a c t e r i a l counts, concentrations of TVBN and TMAN) of the pre-packed, f i s h was found to vary with the type of f i s h involved, besides the nature of the packaging material and the storage temperature. 2.3. Changes under nitrogen modified atmosphere storage. Unlike carbon dioxide, nitrogen, which i s an inert gas, shows no inhibitor y effect on the growth of aerobic microorganisms (Huffman, 1974). Furthermore, i t results i n s l i g h t l y higher anaerobic growth rate than carbon dioxide or oxygen, and i t also does not favor the growth of l a c t i c acid bacteria. 2.4. Multivariate analyses Multivariate analysis i s applicable when one or more independent and one or more dependent variables are being considered simultaneously; each one being considered equally important at the beginning of the analysis (Massart et al., 1978). Each of these measured variables designates i t s own dimension. I t i s beyond the a b i l i t y of man to recognize groupings i n these multidimensional data or to eliminate excessive information. Multivariate techniques can reduce the number of dimensions while retaining the information i n a l l the data. 2.4.1. Factor analysis Factor analysis i s used mainly to aid i n interpretation of complex multivariate data, to study the inter-relationships between the 23 variables. The factor analysis model assumes that the observed variables are manifestations of a number of unobservable factors (Piggott and Sharman, 1986). Variation i n the multivariate data i s summarized into combinations of variables to produce indices that are uncorrelated. These indices are c a l l e d factors. The lack of correlation means the factors are measuring different dimensions of the data. These factors are ranked i n order. The f i r s t factor displays the largest amount of v a r i a t i o n and the second factor displays the second largest amount of the v a r i a t i o n , and so on. The variances i n a factor should be as low as pos-s i b l e , as to be n e g l i g i b l e . In the case where the o r i g i n a l variables are very highly correlated, i t i s quite conceivable that a large number of variables, with most of them measuring similar parameters, can be adequately represented by 2 or 3 factors. 2.4.2. Stepwise discriminant analysis Stepwise discriminant analysis i s a s t a t i s t i c a l technique applied to seek out subsets of variables most useful for discriminating between the treatment groups (Powers and Ware, 1986). The process involves selecting variables, one step at a time. The f i r s t selected variable optimally separates the samples into their predetermined categories, or has the best discrimination power. The next selected variable, with the f i r s t selected variable also taken into consideration, w i l l improve the discrimination the most, and so on. The process continues u n t i l a predetermined l e v e l of significance regarding the separation has been achieved, or a maximum number of steps have been taken. This technique i s especially useful when one needs to screen a large set of response variables i n order to f i n d a smaller subset that w i l l provide good 24 discriminating power. I t i s also useful i n predicting the class to which unknown samples belong. The analysis presents the results i n % correct c l a s s i f i c a t i o n as i n c l a s s i f i c a t i o n matrix and i n Jackknife c l a s s i f i c a t i o n . Unlike the c l a s s i f i c a t i o n matrix, Jackknife c l a s s i f i c a t i o n allocates each individual to i t s closest group without using that individual to help determine the group center (mean). I t i s expected that an observation i s closest to the center of the group where the observation helps to determine that group center. Jackknife c l a s s i f i c a t i o n , therefore, eliminates the bias i n the favour of a l l o c a t i n g the individual to the group that i t r e a l l y comes from (Powers and Ware, 1986; Manly, 1986). MATERIALS AND METHODS 26 3. MATERIALS AND METHODS 3 . 1 . Prawn samples In 1988, two batches of headless shell-on prawns were purchased from a l o c a l seafoods supplier (Murray Fish Co., Ltd., Vancouver, B r i t i s h Columbia) and transported i n waxed paperboard containers layered with crushed ice to the laboratory. The prawns for T r i a l 1 were purchased i n May (past spawning season). The prawns for T r i a l 2 were purchased i n October (approaching spawning season). The majority of the prawns purchased for T r i a l 2 had roe which had to be manually removed. The roe, located at the abdominal part of the prawn body, was p a r t i a l l y enveloped i n between the abdominal carapaces and swimmerets. Removal of the roe was ca r e f u l l y done by inserting a narrow spatula i n between the roe and prawn abdomen and then scraping the roe o f f . However, by doing t h i s , some swimmerets were also torn away and the carapaces were forced open exposing the abdominal tissue. A l l prawns were washed under cold tap water and drained p r i o r to packing. 3.2. Packaging material Packaging material was DUPONT LP 920 (DuPont Canada Ltd., Kingston, Ontario) which i s a laminated p l a s t i c f i l m of polyethylene-polyvinylalcohol-polyethylene. Its properties are shown i n Table 1. I t i s heat-sealable and quite impermeable to gases and water. Bags of 13 cm x 23 cm were made from the LP 920 f i l m . Two hundred grams of the headless shell-on prawns were packed i n each bag. Table 1. Properties*3' of DUPONT LP 920 packaging f i l m N.T.R. = 0.013 cc O.T.R. = 0.05 cc C.T.R. - 0.3 cc M.V.T.R. = 0.3 gm (100 i n2) "1 day"1 atm"1 (100 i n2) "1 day"1 atm"1 (100 i n2) "1 day"1 atm"1 (100 i n2) "1 day"1 atm"1 N.T.R. = Nitrogen Transmission Rate O.T.R. = Oxygen Transmisssion Rate C.T.R. = Carbon dioxide Transmission Rate M.V.T.R. = Moisture Vapour Transmission Rate 28 3.3. Treatments Following the packaging of the prawns, a i r i n the bags was evacuated and replaced with 500 cc of carbon dioxide or nitrogen for the carbon dioxide modified atmosphere packaged (CMAP) samples and nitrogen modified atmosphere packaged (NMAP) samples respectively. A l l bags, except for the control samples, were heat-sealed. The control samples were packed i n the bags l e f t open to a i r . A l l samples were stored at 1°C. 3.4. Sampling plans The day of packing was considered day zero. In T r i a l 1, sampling was done every three days and storage was continued up to day 6 for the control samples and day 18 for the CMAP and the NMAP samples. On each sampling day, 3 bags of each treatment were collected for the determina-t i o n of headspace gas composition, exudate, TSA, and TSN. Another 9 bags of each treatment were transferred into a deep freezer (-18°C) awaiting the other tests: TMAN, K-value, inner tissue pH, sensory evaluations, and Hunter L, a, b values. Sampling, for T r i a l 2, was done every four days and storage was continued up to day 28 for a l l samples. The headspace gas compositions i n the bags were evaluated on the day of packing, day 8, day 16, and day 28 which was the l a s t day of storage. The following analyses were performed i n T r i a l 2: TSA, TSN, SPA, SPN, inner tissue pH, exudate volume, TMAN, TVBN, Hunter L, a, b values, water-soluble proteins (WSP), salt-soluble proteins (SSP), and sensory evaluations. 29 3.5. Tests 3.5.1. Headspace gas compositions in the bags Changes i n headspace gas atmosphere composition within the CMAP and NMAP bags were evaluated. Headspace gas composition i n each of 3 bags of each treatment was evaluated on some selected sampling days by means of a gas chromatographic (GC) method. To analyse the composition of permanent gases of interest which i n -cluded carbon dioxide, oxygen, and nitrogen, a GC system equipped with 2 columns and a switching valve was used. The GC system was a Shimadzu GC-9A (Shimadzu Corp., Kyoto, Japan) with automated programmed temperatures controlled by a microcomputer. A Shimadzu C-R3A Chromatopac (Shimadzu Corp., Kyoto, Japan) data acquisition station was used to record the detector signals and compute peak areas and gas concentration. A thermal conductivity detector (TCD) was used to detect oxygen and nitrogen and a hydrogen-flame ionization detector (FID) was used to detect carbon dioxide. The an a l y t i c a l conditions were: column oven temperature, 50°C; injector temperature, 125°C; TCD temperature, 125°C; FID current, 60 mA; helium c a r r i e r gas flow rate, 25 cc/min. Sampling of the headspace gas i n the bag was done by inserting the needle of an a i r t i g h t glass syringe (with n u l l volume position) through a sil i c o n e septum. The s i l i c o n e septum was made by applying s i l i c o n e seal (Canadian General E l e c t r i c , Toronto, Ontario) onto a Magic Transparent Tape (Scotch Brand, 3M Canada Inc., London, Ontario) and l e f t to dry before putting the tape on to the bag. The tape w i l l allow a good seal with the p l a s t i c f i l m and the s i l i c o n e w i l l form a tight seal after the 30 syringe needle i s removed. The syringe was flushed a few times before c o l l e c t i n g 0.5 cc of the headspace gas sample. The i n j e c t i o n volume was 0.5 cc. The gas sample was f i r s t passed through a porous polymer Porapak Q column (80/100 mesh s i z e , 6 f t ; Water Associates, Waters S c i e n t i f i c Ltd., Missisauga, Ontario) where carbon dioxide was separated. After carbon dioxide was detected, the valve was (a 4 port switching valve, Valco; Supelco Canada Ltd., Oakville, Ontario) switched at an appropriate time to direct the rest of the sample to the second column which was a Molecular Seive 5A (60/80 mesh s i z e , 6 f t ; Supelco Canada Ltd., Oakville, Ontario). Oxygen and nitrogen were then separated. Each run took 7 minutes. Concentrations of carbon dioxide, oxygen, and nitrogen i n the bags were computed. Chromatograms of the headspace gases i n the CMAP and the NMAP bags were shown i n Figures 2 and 3 respectively. 3.5.2. Exudate Determination Three bags from each treatment were used. A corner of the bag was thoroughly swabbed with 90% ethanol, then asep t i c a l l y cut to allow exudate inside the bag to completely drain out of the bag into a graduat-ed cylinder (10 ml) . The exudate from each bag was measured and recorded. The bags had to be cut aseptically because the samples i n these bags were to be used l a t e r for microbiological t e s t s . 3.5.3. Microbiological tests In T r i a l 1, aerobic and anaerobic psychrotrophic microorganisms i n prawns from each of the 3 bags from each treatment of each sampling day were enumerated. F i f t y grams of a sample i n the form of headless s h e l l -3 1 U~i CO CM O O CO CD IT) O CO CM O Time (minutes) Figure 2. Chromatogram of the headspace gases i n the CMAP ba 00 o CO CM CM O (V 00 CD CM O C_> in cu Time (minutes) igure 3. Chromatogram of the headspace gases i n the NMAP bags 33 on prawns were transferred aseptically from the package into a s t e r i l i z e d blender j a r (Fisher S c i e n t i f i c , Ottawa, Ontario). Four hundred and f i f t y m i l l i l i t e r s of 0.1% peptone (Difco, Detriot, Michigan) and 0.5% NaCl were added and the sample was blended to make the f i r s t d i l u t i o n . Appropriate s e r i a l decimal di l u t i o n s of the sample were prepared using the peptone diluent (0.1% peptone, 0.5% NaCl). Each sample d i l u t i o n was then inoculated on to trypticase soy agar (TSA) using a S p i r a l Plater (Spiral System Instrument, Inc., Bethesda, Maryland). Duplicate plates were made. Since the samples originated from cold sea water and were stored under r e f r i g e r a t i o n conditions, 4°C was selected as the incubation temperature. The anaerobic condition was created by introducing a slow stream (15 ml/min) of nitrogen gas, pre-humidified by passing the gas stream through an aqueous solution of 5% ascorbic acid and 0.1% rezasurin to scrub oxygen from the nitrogen gas stream. This anaerobic chamber was made of plexiglass with the design as i l l u s t r a t e d i n Figure 4. The aerobic and anerobic plates were incubated at 4°C for 5 days and 7 days respectively. After the incubation period, the number of colonies formed on each plate was counted and recorded. In T r i a l 2, additional microbiological tests were carried out s p e c i f i c a l l y to monitor the development i n the population of sulfide-producing bacteria i n a l l treatments. The medium used was as described by Sumner and Gorczyca (1984). The inoculated plates were incubated aerobically and anaerobically at 4°C for 10 days. In T r i a l 2, the nitrogen stream was increased to 170 ml/min with the addition of a carbon dioxide stream of 30 ml/min to further exclude the oxygen. 34 r Out Nitrogen gas from gas tank ST > = t \ Flow meter Regulator In Plexiglass Anerobic Chamber 0.1% Rezasurin 5.0% Ascorbic acid Figure 4. Plexiglass anaerobic chamber (38.1 cm x 33.0 cm x 50.8 cm) 35 3.5.4. Determination of pH of prawn inner tissue Each prawn was cut lengthwise into halves. The pH of the inner prawn tissue was measured by using a f l a t t i p pH probe (Accumet pH Meter Model 620, Fisher S c i e n t i f i c , Ottawa, Ontario). The procedure involved one measurement per prawn and f i v e prawns per bag. 3.5.5. K-value determination The K-value of each sample was evaluated based on the high perform-ance l i q u i d chromatographic (HPLC) method of Ryder (1985). 3.5.5.1. Sample preparation From each prawn sample, 25 grams of prawn muscle was blended i n 125 ml of c h i l l e d 0.6 M perchloric acid (BDH Chemical Co., Toronto, Ontario) solution for 60 seconds using an Osterizer (Model Galaxie 8, Sunbeam Appliance Service Co., Vancouver, B.C.), following by centrifugation (3000xg, 10 min, 0°C) i n a Sorvall RC2-B automatic superspeed refrigerated centrifuge (Ivan Sorvall Inc., Newtown, Connecticut). Ten m i l l i t e r s of the supernatant was collected and immediately neutralized to pH 6.5 to 6.8 with 1.0 M potassium hydroxide. After the extract was l e f t at 0°C for 30 minutes, the potassium perchlorate precipitate was removed by f i l t e r i n g the extract through a 0.45 /xm type HA M i l l i p o r e f i l t e r (Millipore®, M i l l i p o r e Ltd., Mississauga, Ontario). The clear f i l t r a t e was diluted to 20 ml and stored at -18°C for l a t e r analysis. A l l solutions used i n this HPLC analysis were prepared by using deionized, d i s t i l l e d water and were f i l t e r e d through a 0.45 fxm type HA M i l l i p o r e f i l t e r p r i o r to inj e c t i n g onto the column. 36 3.5.5.2. HPLC conditions Chromatography was performed on a Spectra-Physics SP 8700 solvent delivery system connected to an SP 8400 variable wavelength detector, and an SP 4100 computing integrator (Spectra-Physics, Santa Clara, CA) . The detector was set at 254 nm. A reverse phase column of Supercosil LC-18 (4.6 mm ID x 25 cm) packed with 5 /im spherical s i l i c a p a r t i c l e s (Supelco Canada Ltd., Oakville, Ontario) was used for the chromatographic analysis. Column temperature was maintained at 30°C with a Controller Model LC-22 and a Column Heater Model LC-23 (Bioanalytical System Inc., West Lafayette, Indiana). A 10 sample loop was used on the i n j e c t o r . An MPLC RP-8 Spheri-10 RP-GU guard column (Brownlee Labs Inc., Santa Clara, CA) was placed before the Supercosil LC-18 column. This guard column (4.6 mm ID x 3 cm) was packed with t o t a l l y porous 10 m^ Spheri-10. The guard column was changed often. An SP 4100 computing integrator was used to calculate individual and t o t a l peak areas. A chart speed of 1 cm/minute, an attenuation of 4 with a range of 0.04, a default peak width value of 6, and a default peak threshold value of 12 were used throughout the study. The mobile phase was a buffer solution of 0.04 M potassium dihydro-gen orthophosphate and 0.06 M dipotassium hydrogen orthophosphate. Both potassium dihydrogen orthophosphate and dipotassium hydrogen phosphate were of HPLC grade (BDH Chemical Co., Toronto, Ontario). An i s o c r a t i c mobile phase was run at a flow rate of 2 ml/minute. The mobile phase was f i l t e r e d through a 0.45 type HA M i l l i p o r e f i l t e r (Miilipore®, M i l l i -pore Ltd., Mississauga, Ontario) before use and was prepared d a i l y . 37 Water and methanol were used to clean the column. The mobile phase, water, and methanol were degassed for 15 min prior to use. During the elution or the cleaning process, solutions were maintained i n a degassed state with a slow stream of helium gas. 3 . 5 . 5 . 3 . Reproducibility of the HPLC analysis The rep r o d u c i b i l i t y of the HPLC analysis was examined with a stand-ard solution consisting of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), inosine monophosphate (IMP), inosine (HxR), 5-bromouracil (the internal standard used for the quantification of the K-value), and hypoxanthine (Hx) (Sigma® Chemical Company, St.Louis, Missouri). The standard solution was injected onto the column 3 times a day for 5 days. A chromatogram of the nucleotide standard working solution i s i l l u s t r a t e d i n Figure 5. The results are shown i n Table 2. 3 . 5 . 5 . 4 . Internal standard 5-Bromouracil was chosen to be the internal standard i n order to quantify the K-value. Chromatogram of the internal standard 5-bromouracil stock solution i s i l l u s t r a t e d i n Figure 6. The 5-bromouracil had a retention time of 8 minutes. However, i t was found to produce another tiny peak with a retention time of 3.4 minutes, the same retention time for the ATP. The minute 8 peak showed a s i g n i f i c a n t l y greater area count than the minute 3.4 peak which seemed to be either a degraded component or some impurities. Effect of this tiny peak on the quantification of the K-value was investigated. Three different solu-tions were used. Time (minutes) Figure 5. Chromatogram of the nucleotide standard working solut 39 Table 2. The rep r o d u c i b i l i t y of the HPLC column. Retention Time (minutes) IMP ATP ADP AMP Hx I.S. HxR Dayl 2. .43 3. .31 3. .69 4. .24 4. .96 8. .06 17. .32 Day2 2 .61 3. .69 4. .12 4. .72 5. .56 9. .05 19, .61 Day3 2. .55 3. .53 3. .95 4. ,53 5. .33 8. .71 18, .34 Day4 2 .58 3. .58 4. .00 4. .60 5. .43 8. .87 18. .80 Day5 2. .55 3. .51 3. .93 4. .53 5. .35 8. .72 18. .41 Mean 2. .54 3. .52 3. .94 4. .52 5, .33 8. .68 18. .50 s.d. 0. .06 0. ,12 0. .14 0. ,16 0. .20 0. .33 0. .74 c. v. 2, .41 3. .52 3. .57 3. .49 3, .76 3. .85 4. .01 Area count x 1000 IMP ATP ADP AMP Hx I.S. HxR Dayl 78. .46 120. ,98 98. .83 142. .67 192. ,69 21. .63 199. ,22 Day2 79. .58 127. .19 102. .02 148. .98 201. .50 22. .34 206. ,95 Day3 79, .08 125. .23 102. .11 147, .23 201. .38 22. .47 205. .90 Day4 80. .35 127. .17 103. .84 149, .45 204. .14 22. .47 207. .99 Day5 81. .37 126. .30 103. .82 149, .43 203. .96 22, .52 207. .54 Mean 79. .77 125. .37 102. .12 147, .55 200. .73 22. .29 205, .52 s.d. 1. .01 2. .31 1. .83 2. 57 4. .19 0. .33 3. .23 ; c. v 1. .27 1. .84 1. .79 1 .74 2. .09 1. .50 1. .57 Figure 6. Chromatogram of the internal standard 5-bromouracil stock solution 41 Solution A consisted of only the standard solution (a mixture of ATP, ADP, AMP, IMP, Hx, and HxR). Solution B consisted of the standard solution (3 ml) with the addition of 100 fil 5-bromouracil (Sigma® Chemical Company, St.Louis, Missouri). Solution C was made by adding 100 fil 5-bromouracil to 3 ml buffer solution (eluting medium). The results (Table 3) showed that there was no si g n i f i c a n t difference i n the K-values between solution B and solutions A and C. Quantification of the K-value by this HPLC method was based on the stock internal standard method. The o r i g i n a l concentration of 5-bromouracil was made (46 mg/50 ml). In each sample extract or standard solution of 2 ml, 100 fil of this stock solution of the internal standard was added. This would give the f i n a l concentration of the 12 nmole of internal standard i n a 10 fil i n j e c t i o n . By knowing the weight of each component i n the standard solution, the concentration of the stock internal standard, the d i l u t i o n , and the area counts of a l l components involved, the concentration of each compo-nent i n the standard solution was determined. With this information the K-value was calculated. 3.5.5.5. Reproducibility of the extraction Three extracts were made from one prawn sample. The extracts were then analysed for the K-values. The results are shown i n Table 4. 3.5.6. Trimethylamine-nitrogen determination Trimethylamine-nitrogen (TMAN) leve l i n each sample was evaluated with the colorimetric method of A.O.A.C. 1984 (sections 18.031-18.033). Table 3. The K values of the nucleotide standard solution with and without the area count of the small unknown peak (n=3) Area Count Solution A Solution B Soluiton C Solution B-C IMP 51755. .67 51369. .67 51369. .67 ATP 79538. .33 78628, .67 *1298. .00 (77330. .67) ADP 62171. .67 60368. .00 60368. .00 AMP 104370. .67 102337 .00 102337. .00 Hx 59393. .00 57368, .67 57368. .67 I.S. (24722. .67) 23609, .67 24722. .67 23609. .67 HxR 56428. .67 55336, .00 55336. .00 % K value 33. .87 33, .64 33. .74 Mean 33.75 Standard deviation 0.09 Coefficient of variance % 0.28 * •= The small unknown peak Solution A = The nucleotide standard solution pluses the area count of the internal standard from solution C Solution B = The nucleotide standard solution with the intern a l standard Solution C = The internal standard i n the buffer solution (mobile phase) Solution B-C = The area count of the small unknown peak from solution C was deducted from the area count of ATP i n solution B Table 4. The reproducibility of the extraction for K value determination Area Count Extractl Extract2 Extract3 Mean s. d. % c. . v. IMP 73983. .33 79452. .67 77017, .50 76817. .83 2237. .31 2. .91 ATP 0. .00 0. .00 0. .00 0. .00 0. .00 0. .00 ADP 9075. .67 9680. .33 9267, .00 9341. .00 252. .34 2. .70 AMP 41034. .67 48734, .00 38439 .00 42735. .89 4371. .68 10. .23 Hx 21575. .33 22455. .33 24265, .00 22765 .22 1119. .70 4. .92 I.S. 21781. .33 21429, .33 21343, .00 21517, .89 189. .59 0. .88 HxR 39600. ,00 41320. .00 40023, .50 40314. .50 731. .72 1. .82 % K value 37. .95 36. .75 38. .76 37, .82 0. .83 2. .18 44 One hundred grams of prawn muscle were blended for 1 min i n 200 ml 7.5% tri c h l o r o a c e t i c acid with a Waring blender (Fisher S c i e n t i f i c , Ottawa, Ontario) which was connected to a variable autotransformer (Sta-co, Inc., Dayton, Ohio; type 2PF 1010, input 120 KVA 1.4, output 0-120/140, Amp. 10, Freq. 50/60) monitored at 80 on the scale. The macerate was centrifuged (3000xg, 10 minutes, 0°C) i n a Sorvall RC2-B automatic superspeed refrigerated centrifuge (Ivan Sorvall Inc., Newtown, Connecticut). The supernatants were kept at -18°C pr i o r to further analyses. 3.5.7. Total volatile basic nitrogen determination The steam d i s t i l l a t i o n method of Malle and Tao (1987) was used to determine levels of TVBN i n the samples. One hundred grams of prawn muscle were homogenized for 1 min i n 200 ml 7.5% tr i c h l o r o a c e t i c acid with a Waring blender (Fisher S c i e n t i f i c , Ottawa, Ontario) which was connected to a variable autotransformer (Staco, Inc., Dayton, Ohio; type 2PF 1010, input 120 KVA 1.4, output 0-120/140, Amp. 10, Freq. 50/60) monitored at 80 on the scale. The homogenate was centrifuged (4000xg, 5 minutes, 0°C) i n a Sorvall RC2-B automatic superspeed refrigerated centrifuge (Ivan Sorvall Inc., Newtown, Connecticut). The supernatant was f i l t e r e d through a Buchner funnel using a Whatman No.3 f i l t e r paper. The supernatants were kept at -18°C awaiting further analyses. 3.5.8. Sensory evaluations A group of 7 panelists were a l l trained to d i f f e r e n t i a t e the follow-ing q u a l i t i e s : odour and colour of raw prawn meat, and colour, odour, 45 flavour, and texture of cooked prawn meat. There were 5 tr a i n i n g ses-sions i n 5 consecutive weeks. The panel was trained with fresh prawns and prawns that were kept at re f r i g e r a t i o n temperature to the point of spoilage. In each session, changes i n the characteristics of fresh prawns and refrigerated prawns i n both raw and cooked forms were compared and discussed. A l l changes i n the characteristics of prawns used as a guide i n th i s study were reported by Varga et a l . , (1972) as i l l u s t r a t e d i n Table 5. Sensory evaluation was done by using a 9-point hedonic scale: 1 = d i s l i k e extremely to 9 = l i k e extremely as shown i n Figures 7 to 9. A score of 9 indicates excellent quality prawns which i s pink or orange-pink body; fresh seaweedy odour; sweet and r i c h flavour; firm and e l a s t i c texture. A score of 5 i s assigned the lower l i m i t of acceptable q u a l i t y . I t describes prawns with faded colour and yellowish-green staining; s l i g h t l y musty, s l i g h t l y s t a l e , and/or very s l i g h t l y ammoniacal odour; s t a l e , sweet, and/or s l i g h t l y b i t t e r aftertaste; and s o f t , mushy, and limp texture. Scores below 5 were considered to be of unacceptable q u a l i t y . The panel was instructed that i f any sample was doubtful, they had a choice of not tasting that sample and to taste that sample was a voluntary action. In T r i a l 1, seven panelists participated in, the sensory evaluation i of prawn samples. In T r i a l 2, s i x out of the seven panelists were available. Samples were presented to the panel i n random order. A l l prawn samples used i n the sensory evaluation were manually peeled. For the evaluation of odour, flavour, and texture of the prawn samples, two prawns were put i n a aluminum f o i l cup and t i g h t l y covered with an aluminum f o i l sheet. These cups were placed on a baking tray and Table 5. Characteristic changes of pink prawns during storage A p p e a r a n c e O d o u r F I a v o u r T e x t u r e 10 Pink, Orange-pink body Fresh, characteristic odour, seaweedy Full fresh flavour, sweet, very rich, characteristic of fresh shrimp Firm, elastic 9 Pale pink, slightly discolouration on heads Fresh, seaweedy Slight loss of flavour Fi rm, elastic a Slightly faded pigment, greenish-yellow liver staining Slightly seaweedy Sweetish, lingering sweet aftertaste Firm, slight loss of elasticity 7 Faded pigment, brownish discolouration on heads, greenish-yellow liver staining Neutral to very slightly stale Slightly sweetish to neutral Firm, slightly elastic, some limpness 6 Faded pigment, brown discolouration on heads, yellowish-green liver staining slightly state, fishy Neutral to slightly stale, sweetish Slight softness, loss of elasticity 5 Faded pigment, brown discolouration on heads, yellowish-green liver staining Slightly musty, siightly stale, very slightly ammonical Stale, sweet, slightly bitter aftertaste Soft, mushy, limp 4 Very faded pigment, yellowish-green liver stafning, black discolouration on heads Strongly ammonical, stale, musty Stale, sweet, bitter, bitter aftertaste Mushy, limp 3 to 0 Very faded pigment, very badly discoloured, very badly liver stained Strongly ammonical, putrid Very bitter, objectionable, putrid Mushy 4 8 Figure 7. Sensory evaluation score sheet for cooked prawn meat odour, flavour, and texture scores NAME DATE Please evaluate the COOKED prawn samples by checking your ratings on the i r odour, f l a v o r , and texture. Commenting on each parameter i s encouraged. Sample code | Comments : Odour Flavor Texture | Like extremely | Like very much | Like moderately | Like s l i g h t l y | Neither l i k e nor d i s l i k e | Di s l i k e s l i g h t l y , | Di s l i k e moderately | Di s l i k e very much | Di s l i k e extremely | Sample code | Comments : Odour Flavor Texture | Like extremely | Like very much | Like moderately , | Like s l i g h t l y | Neither l i k e nor d i s l i k e | Di s l i k e s l i g h t l y | Di s l i k e moderately | Di s l i k e very much _ _ _ _ _ | Di s l i k e extremely | Sample code | Comments : Odour Flavor Texture | Like extremely j | Like very much | Like moderately | Like s l i g h t l y • | Neither l i k e nor d i s l i k e | Di s l i k e s l i g h t l y | D i s l i k e moderately , | Di s l i k e very much 1 | Di s l i k e extremely | 50 Figure 8. Sensory evaluation score sheet for cooked prawn meat colour score 51 NAME DATE Please evaluate the COOKED prawn samples by checking your ratings on the i r colour. Commenting on each parameter i s encouraged. Sample code Sample Code Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e D i s l i k e s l i g h t l y D i s l i k e moderately D i s l i k e very much Di s l i k e extremely Sample code Sample Code Like extremely Like very much "  Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e •  Di s l i k e s l i g h t l y D i s l i k e moderately D i s l i k e very much Di s l i k e extremely Sample code Sample Code Like extremely Like very much Like moderately Like s l i g h t l y _ Neither l i k e nor d i s l i k e D i s l i k e s l i g h t l y D i s l i k e moderately D i s l i k e very much j Di s l i k e extremely Comments: 52 Figure 9. Sensory evaluation score sheet for raw prawn meat odour and colour scores NAME DATE Please evaluate the RAW prawn samples by checking your ratings on the i r colour and odour. Commenting on each parameter i s encouraged. Sample code Sample code Colour Odour Colour Odour Like extremely Like very much - Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e D i s l i k e s l i g h t l y D i s l i k e moderately - Di s l i k e very much Di s l i k e extremely Sample code Sample code Colour Odour Colour Odour Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e D i s l i k e s l i g h t l y D i s l i k e moderately D i s l i k e very much Di s l i k e extremely Sample code Sample code Colour Odour Colour Odour Like extremely Like very much Like moderately Like s l i g h t l y Neither l i k e nor d i s l i k e D i s l i k e s l i g h t l y D i s l i k e moderately D i s l i k e very much Di s l i k e extremely Comments: 54 put i n a conventional oven preheated at 200°C. Cooking was continued for 25 min. Cooked samples were kept warm at 60°C for serving. For evaluation of the colour of the prawn samples, two prawns were put i n a clear glass bowl and then t i g h t l y covered with an aluminum f o i l sheet. These bowls were placed on a baking tray. Cooking was carried out i n the same manner as above. Figure 10 showed the temperature gradient' at the center of the preheated (200°C) conventional oven after i t was opened and then closed (simulating that samples were being loaded). The temperature gradient inside the edible prawn tissue during cooking i s shown i n Figure 11. I t was shown that within 15 min after loading, the temperature inside the edible prawn tissue was maintained at approximately 100°C. For the odour and colour of the raw prawn samples, two prawns were put i n a clear glass bowl and then t i g h t l y covered with an aluminum f o i l sheet. These bowls were kept i n a 4°C cold room p r i o r to the panel te s t s . For each t e s t , samples were presented to each panelist i n random or-der to balance out any order effects that might occur. The prepared samples were placed on a white paper plate before serving to each panel-i s t . F i r s t , the evaluations of odour, flavour, and texture of the cooked prawn samples were done under the red-masked, l i g h t . Second, the •I evaluation of colour of the cooked prawn samples; and t h i r d , the evaluation of odour and colour of raw prawn samples were carried out under normal white l i g h t . Room temperature water with a s l i g h t lemon taste and unsalted soda crackers were served so each panelist could cleanse the i r mouth between samples. 55 205 0 5 10 15 20 25 30 Minutes after loading Figure 10. Temperature gradient at the center of the preheated (200°C) conventional oven Figure 11. Temperature gradient inside the edible prawn tissue during cooking at 200°C 57 3.5.9. Hunter L, a, b values The colours of raw, headless, shell-on prawns were measured by an objective method based on the Hunter opponent-colour scales: L, a, b values for lightness/darkness, redness/greenness, and yellowness/blueness scales respectively. Measurement was performed using a Hunterlab spectrocolorimeter (Labscan I I IBM 0°/45°, Hunter Associates Laboratory, Inc., Reston, V i r g i n i a ) . A single prawn was placed on a new s t e r i l i z e d disposable p l a s t i c p e t r i d i s h (Fisherbrand, 100x15mm Standard, Fisher S c i e n t i f i c Company, Ottawa, Ontario). An aperture size of 1.0 cm with a single reading per prawn was used throughout the study. The Hunter opponent-colour values were determined on the same day as the sensory evaluation tests were conducted. 3.5.10. Water-soluble and salt-soluble protein determinations Water-soluble and salt-soluble proteins were extracted according to the procedure used by Cobb and Vanderzant (1971). Water-soluble proteins were extracted from 25 gm prawn tissue using 50 ml d i s t i l l e d water. To extract salt-soluble proteins, the residue was blended with 50 ml of pH 7.2 buffer which consisted of 0.45 M KC1, 0.0157 M Na2HP04, and 0.0031 M KH2P04. The protein nitrogen levels of the extracts were determined using BCA Protein Assay Reagents (Pierce Chemical Company, Rockford, I l l i n o i s ) . Incubation at 60°C for 30 minutes was used throughout the study. 3.6. Statistical analyses Factor analysis and stepwise discriminant analysis were performed on an Amdahl 470 V/8. Both analyses are i n the BMDP (Biomedical Programs, 58 BMDP S t a t i s t i c a l Software Inc., 1985). Factor analysis and stepwise discriminant analysis were performed on selected variables from the data of T r i a l 2 (n=720) . The data input i s as shown i n Figure 12 and was cal l e d "multivariate data". The multivariate data included the data of 20 variables obtained from each of 3 bags of each treatment (the control, the CMAP, and the NMAP assigned as treatment number 1, 2, and 3 respectively) during the 12 day storage period (4 sampling days). Only the data obtained up to day 12 were analysed because by day 12, prawns from a l l treatments were of unacceptable quality based on sensory point of view (overall sensory scores). The data was rearranged i n such a way that time function could be included i n the analyses with a l l required data for each variable presented as one data subset. For factor analysis, program P4M i n the BMDP was applied to the multivariate data. Direct quartimin rotation was used to rotate factors so that the variance of the squared loadings was maximized within variables. This rotation method si m p l i f i e s variables by producing one or more large loadings and the rest as near to zero as possible. For stepwise discriminant analysis, program P7M i n the BMDP programs was applied to the multivariate data. In this study, the purpose of using the stepwise discriminant analysis was to obtain equations that aid the c l a s s i f i c a t i o n of these prawn products according to their degree of freshness or spoilage. TMAN concentration was selected as the discriminant variable because i t i s the most suitable freshness/spoilage indicator for the prawns i n this study. This was because while other spoilage compounds were the results of ba c t e r i a l and tissue enzyme a c t i v i t i e s , TMA was mainly produced by bacteria during storage and i t s Figure 12. Multivariate data input format MAP Bag Day Drip pH TSA TSN SPA SPN TMAN TVBN WSP SSP L a b 0.0 7.40 6.35 5.97 1.70 0.40 0.06 0.38 30.17 34.10 37.28 12.04 12.37 0.0 7.60 6.05 6.10 2.16 1.00 0.07 0.55 26.79 43.86 37.89 12.52 11.88 0.0 7.34 5.24 4.99 1.48 0.88 0.04 0.61 26.27 39.68 37.07 11.66 11.83 0.0 7.40 6.35 5.97 1.70 0.40 0.06 0.38 30.17 34.10 37.28 12.04 12.37 0.0 7.60 6.05 6.10 2.16 1.00 0.07 0.55 26.79 43.86 37.89 12.52 11.88 0.0 7.34 5.24 4.99 1.48 0.88 0.04 0.61 26.27 39.68 37.07 11.66 11.83 0.0 7.40 6.35 5.97 1.70 0.40 0.06 0.38 30.17 34.10 37.28 12.04 12.37 0.0 7.60 6.05 6.10 2.16 1.00 0.07 0.55 26.79 43.86 37.89 12.52 11.88 0.0 7.34 5.24 4.99 1.48 0.88 0.04 0.61 26.27 39.68 37.07 11.66 11.83 8.0 7.31 7.87 7.95 4.24 4.03 3.06 1.46 38.33 41.65 38.32 10.73 11.68 5.6 7.33 7.47 7.49 4.19 4.15 2.83 1.25 39.74 47.27 37.18 10.95 11.56 5.5 7.23 7.55 7.70 4.25 4.18 1.61 1.17 36.68 45.73 41.18 13.29 12.96 7.0 6.50 5.36 5.43 2.08 1.40 2.90 1.08 29.45 55.99 40.54 13.17 12.60 11.0 6.41 6.03 6.09 2.21 2.68 2.24 1.07 34.00 53.47 38.17 12.77 11.58 6.5 6.44 6.12 6.15 2.99 3.07 1.20 0.88 31.09 54.41 37.11 11.95 12.10 7.7 7.13 7.58 7.66 4.27 4.47 4.08 1.17 41.04 52.70 38.95 14.16 11.65 7.8 7.10 7.59 7.63 4.21 4.27 3.66 1.06 42.44 54.34 38.00 14.21 12.21 5.2 7.06 7.17 7.21 4.13 4.07 2.71 1.15 48.36 46.73 39.71 13.45 13.38 4.9 7.89 8.48 8.58 5.10 4.00 13.60 0.96 45.87 43.71 37.97 11.99 13.39 4.2 7.65 8.58 8.64 4.97 4.10 12.00 0.69 43.14 40.41 38.12 11.32 12.78 3.7 7.74 8.47 8.54 4.89 4.18 11.90 0.58 43.31 47.07 38.28 11.07 13.30 9.2 6.64 6.46 7.05 3.28 2.99 5.85 0.37 25.39 50.65 40.73 12.96 12.74 8.7 6.49 6.17 6.20 3.32 2.10 1.90 0.24 22.15 44.34 39.20 13.04 12.52 11.9 6.57 7.22 7.32 3.34 2.95 3.30 0.53 26.02 48.20 40.63 15.48 13.32 3.2 7.39 7.94 7.94 5.12 4.68 32.05 0.91 40.75 63.05 39.53 14.34 11.85 2.3 7.40 7.76 7.89 5.08 4.94 13.80 0.63 35.18 55.26 39.63 15.32 11.98 6.4 7.31 8.01 8.07 5.12 5.12 27.15 0.78 41.38 52.47 40.27 16.23 12.50 5.8 8.04 9.95 9.92 6.60 6.30 23.76 1.02 106.01 21.30 35.77 10.60 11.85 5.6 8.12 10.06 9.93 6.98 6.92 28.38 1.21 102.01 26.24 36.05 11.64 12.88 4.9 8.11 9.83 9.82 6.72 6.51 28.93 1.17 103.62 30.16 35.54 10.27 12.54 13.1 6.69 8.03 8.04 5.33 4.54 26.51 0.72 35.16 54.30 41.80 13.95 12.66 7.8 6.67 7.9 8.03 5.12 4.70 10.34 0.42 36.76 50.50 40.81 13.84 14.01 2.4 7.14 8.38 8.37 5.34 4.65 8.91 0.55 46.13 59.52 43.56 14.47 12.79 6.1 7.46 9.33 8.86 5.13 5.15 41.25 1.31 52.77 62.92 40.37 13.92 12.56 3.8 7.47 9.19 8.93 4.99 5.45 37.07 1.00 60.61 55.73 39.68 14.26 11.98 4.5 7.51 9.19 8.95 4.86 5.05 52.80 1.39 53.37 52.09 39.62 14.31 11.61 1 1 0 1 2 0 1 3 0 2 1 0 2 2 0 2 3 0 3 1 0 3 2 0 3 3 0 1 1 4 1 2 4 1 3 4 2 1 4 2 2 4 2 3 4 3 1 4 3 2 4 3 3 4 1 1 8 1 2 8 1 3 8 2 1 8 2 2 8 2 3 8 3 1 8 3 2 8 3 3 8 1 1 12 1 2 12 1 3 12 2 1 12 2 2 12 2 3 12 3 1 12 3 2 12 3 3 12 OVERALL 6.50 6.50 6.50 6.50 6.50 6.50 .50 .50 .50 .67 ,67 .67 .47 ,47 ,47 5.75 5.75 5.75 4.45 4.45 4.45 5.04 5.04 5.04 5.31 5.31 5.31 3.42 3.42 3.42 4.81 4.81 4.81 4.43 4.43 4.43 RC 6.33 6.33 6.33 6.33 6.33 6.33 6.33 6.33 6.33 6.17 6.17 6.17 7.17 7.17 7.17 6.00 6.00 6.00 4.50 4.50 4.50 5.83 5.83 5.83 6.00 6.00 6.00 17 17 17 83 83 83 5.40 5.40 5.40 RO 7.33 7.33 7.33 7.33 7.33 7.33 7.33 7.33 7.33 6.67 6.67 6.67 7.00 7.00 7.00 6.00 6.00 6.00 4.00 4.00 4.00 5.83 5.83 5.83 4.67 4.67 4.67 2.33 2.33 2.33 6.00 6.00 6.00 3.50 3.50 3.50 CC 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 5.67 5.67 5.67 4.50 4.50 4.50 6.83 6.83 6.83 5.33 5.33 5.33 4.17 4.17 4.17 6.67 6.67 6.67 3.83 3.83 3.83 4.50 4.50 4.50 6.00 6.00 6.00 CO 5.33 5.33 5.33 5.33 -5.33 5.33 5.33 5.33 5.33 4.00 4.00 4.00 3.83 3.83 3.83 4.50 4.50 4.50 2.83 2.83 2.83 4.00 4.00 4.00 4.17 4.17 4.17 2.33 2.33 2.33 3.50 3.50 3.50 2.17 2.17 2.17 CF 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 5.17 5.17 5.17 5.17 5.17 5.17 5.17 5.17 5.17 3.83 3.83 3.83 5.60 5.60 5.60 5.00 5.00 5.00 2.50 2.50 2.50 4.67 4.67 4.67 4.50 4.50 4.50 CT 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.67 6.33 6.33 6.33 5.17 5.17 5.17 6.00 6.00 6.00 6.20 6.20 6.20 4.80 4.80 4.80 5.33 5.33 5.33 5.33 5.33 5.33 6.33 6.33 6.33 5.00 5.00 5.00 ON o 61 concentration increased gradually i n a considerably steady rate throughout the 12 days storage. Moreover, i t s association with fishy odour and off-odours i n seafood products makes i t suitable to be used to indicate the quality of seafood products. Based on studies by Cas t e l l et a l . , (1958) and Castell and Greenough (1958), TMAN concentration was used to c l a s s i f y f i s h quality into three grades. Grade 1 f i s h or prime quality f i s h contained 0 to 1 mg TMAN/100 gm muscle. Grade 2 f i s h or marketable f i s h contained 1 to 5 mg TMAN/100 gm muscle. Grade 3 f i s h or unacceptable f i s h contained more than 5 mg TMAN/100 gm muscle. In some sectors of Japanese and Australian markets, 5 mg TMAN/100 gm muscle tissue i s also used as the acceptability l i m i t for shrimp (Montgomery et a l . , 1970). In this study, three ranges of TMAN concentration were used to establish outpoints for separation of the prawn data i n the stepwise discriminant analysis. These ranges were TMAN concentrations up to 1.00, 1.01 to 5.00, and more than 5.00 mg TMAN/100 gm of prawn tissue respectively. These three ranges of TMAN concentration represent good, acceptable, and unacceptable quality respectively. Obviously, to evaluate the quality of a highly perishable food prod-uct, the tests should be simple, r e l i a b l e , less time-consuming, inexpensive, and the number of the tests should also be minimized. To achieve t h i s , f i r s t , variable pairs that had high correlations were selected from the correlation matrix obtained by the factor analysis. Second, variables that gave similar information were eliminated. After the stepwise discriminant analyses were run on the data of these selected variables, only the variable(s) that produced highest percent correct c l a s s i f i c a t i o n of these prawn samples were the f i n a l s e l e c t i o n . 62 Analysis of variance (ANOVA), Tukey's te s t , student's paired T-test, and Friedman two-way ANOVA were performed using SYSTAT program (Systat Inc., 1988). In the SYSTAT program, by running a Tukey's t e s t , an ANOVA was automatically included and an ANOVA table was produced. These tests were done on data of each treatment and data of each sampling day. Tukey's test determines the groups of data that were and were not s i g n i f i c a n t l y d i f f e r e n t . A student's paired T-test was done on microbiological data to determine i f there was any s i g n i f i c a n t difference between aerobic and anaerobic microbial populations. Friedman two-way ANOVA was applied to sensory scores given by each panelists. This s t a t i s t i c a l analysis ranks the sensory scores p r i o r to proceeding with the ANOVA thus overcoming the va r i a t i o n that was l i k e l y to happen when each pane l i s t , even when the same measurment method was used, the scale was not quite exactly the same to a l l panelists. Day to day veriations were also overcome by this method. Another two-way ANOVA (O.Mahony, 1986) was applied to the sensory data from each panelist to determine i f there was any si g n i f i c a n t difference among judges. 63 4. RESULTS 64 4. RESULTS 4.1. T r i a l 1 4.1.1. Headspace gas composition A s l i g h t increase i n carbon dioxide concentration i n the NMAP bags occurred during storage (Figure 13). Changes i n nitrogen concentration i n the headspace of the bags were proportional to the changes of the carbon dioxide concentration. Figure 14 shows that at day 0, most but not a l l of the a i r i n the bag was evacuated before backflushing with carbon dioxide. I t shows that carbon dioxide concentration i n the CMAP bags dropped soon after the storage started and l a t e r slowly increased. Carbon dioxide concentrations i n the CMAP bags differe d s i g n i f i c a n t l y (P<0.01) during storage with that of day 3 being the lowest and s i g n i f i c a n t l y different from the rest. Carbon dioxide concentrations i n the NMAP bags also differ e d s i g n i f i c a n t l y (P<0.01) during storage with carbon dioxide concentration of each sampling day being s i g n i f i c a n t l y different from the other days. 4.1.2. Exudate formation The highest exudate volume occurred i n CMAP prawns followed by the NMAP and the control prawns respectively (Figure 15). By day 18, the amount of exudate formed i n the CMAP prawns was about twice that formed i n the NMAP prawns. No si g n i f i c a n t (P>0.01) changes i n exudate volume during 6 days storage were found i n each treatment (Table 6) . During 18 days storage, however, change i n the exudate volume was s i g n i f i c a n t 65 C (D CL 100 80 60 40 20 I f 96136 0 0.5SQ: 2Jl7( 3_3 i i : 90.80 9.04 DayO Day3 Day9 Day18 Figure 13. T r i a l 1: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the NMAP bags stored at 1°C 66 r C <U Q_ 100 80 60 40 20 0 93.11 1 i 1 88.09 88.30 81.01 5.74 . in 6.77 .Z i.1 f .74 mi 11.59 m DayO Day3 Day9 Day18 Figure 14. T r i a l 1: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the CMAP bags stored at 1°C 67 0 3 . 6 9 12 15 18 Days of Storage Figure 15. T r i a l 1: Exudate volumes of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C Table 6. F - s t a t i s t i c s (a) from ANOVA on data of T r i a l 1 I. Data of 6 days storage Variables (b) Control CMAP NMAP Exudate (c) 0.585 0 .195 0.567 pH 1.666 35 .299 ** 0.499 TSA 293.742 ** 15 .696 ** 106.344 ** TSN 326.853 ** 19 .339 ** 178.23 ** TMAN 46.240 ** 2 .643 12.569 ** IMP 11.397 ** 3 .564 11.733 ** ADP 3.611 5 .543 * 0.736 AMP 65.379 ** 26 .668 ** 26.596 ** Hx 12.152 ** 7 .467 * 28.812 ** HxR 1.544 0 .149 0.754 K-value 29.766 ** 119 .973 ** 161.374 ** L 0.007 17 .164 ** 12.659 ** a 0.283 17 .919 ** 5.314 b 5.864 * 13 .363 ** 10.711 ** II. Data of 18 days storage Variables CMAP NMAP Exudate 3 .386 * 2.401 pH 18 .418 ** 6.825 ** TSA 69 .798 ** 70.687 ** TSN 65 .036 ** 141.902 ** TMAN 37 .792 ** 146.387 ** IMP 11 .834 ** NV (d) ADP 5 .498 ** 6.238 ** AMP 49 .399 ** 52.140 ** Hx 12 .691 ** 31.381 ** HxR 1 .618 24.635 ** K-value 71 .106 ** 183.839 ** L 9 .055 ** 11.231 ** a 9 .235 ** 16.285 ** b 9 .325 ** 8.667 ** (a) * = s i g n i f i c a n t at 5% l e v e l ** = s i g n i f i c a n t at 1% l e v e l (b) See Abbreviation L i s t (c) Only data of day 3-day 6; no exudate on day 0 (d) Data became zero during storage Table 7. F-statistics (a) from ANOVA on data of individual sampling day of T r i a l 1 (b) Variables (c) Day3 Day6 Day9 Exudate 16.405 ** 3.521 8.204 PH 59.445 ** 51.754 ** 169.615 ** TSA 69.834 ** 475.244 ** 409.862 ** TSN 152.911 ** 342.504 ** 139.754 ** TMAN 1.518 1.741 31.087 ** IMP 1.075 3.329 26.370 ** ADP 12.908 ** 2.925 3.357 AMP 0.892 3.574 3.019 Hx 1.234 0.404 4.384 HxR 2.073 2.417 79.849 ** K-value 0.569 0.229 9.071 * L . 7.655 * 7.372 * 0.637 a 2.213 13.927 ** 8.269 b 1.711 1.404 11.158 Variables Dayl2 Dayl5 Dayl8 Exudate 0.307 5.647 28.735 ** PH 336.538 ** 133.225 ** 44.569 ** TSA 96.571 ** 11.521 * 15.556 * TSN 97.595 ** 63.034 ** 12.956 * TMAN 25.181 ** 32.107 ** 224.210 ** IMP 2.999 5.117 3.857 ADP 0.350 0.508 4.701 AMP 0.131 0.164 0.628 Hx 20.172 * 7.329 9.899 * HxR 2.245 6.922 20.555 * K-value 2.333 4.505 4.155 L 1.411 0.186 0.212 a 9.136 ** 4.571 ** 3.058 b 0.998 3.228 0.559 (a) * = significant at 5% level ** = significant at 1% level (c) See Abbreviation List (b) For day 3 and day 6, compared control, CMAP, and NMAP For day 9 to day 18, compared CMAP and NMAP 70 (P<0.05) i n the CMAP prawns. I t was on day 3 that differences i n exudate volume of prawns from these three treatments were s i g n i f i c a n t (Table 7). 4.1.3. Tissue pH The prawns at the time of packing had a pH of about 7.2 (Figure 16). The pH of the control prawns increased during storage. After a s l i g h t i n i t i a l drop, the tissue pH of the the NMAP prawns s l i g h t l y increased. After day 9, the pH of the NMAP prawns remained i n the range of 7.3 to 7.4 u n t i l the end of storage. The tissue pH of the CMAP prawns dropped to 6.6 within 3 days of storage and then increased s l i g h t l y to 6.9 by the l a s t day (day 18) of storage. There were no si g n i f i c a n t (P>0.05) changes i n tissue pH i n the control and the NMAP prawns during the f i r s t 6 days of storage. On the contrary, the changes i n the tissue pH of the CMAP prawns were si g n i f i c a n t during the f i r s t 6 days of storage. Significant changes i n the tissue pH were found during the 18 days storage of the CMAP and the NMAP prawns (P<0.01, Table 6). The tissue pH values of the prawns from three treatments were s i g n i f i c a n t l y different on each sampling day throughout the 18 days storage (P<0.01, Table 7). 4.1.4. Microbiology The highest t o t a l psychrotrophic b a c t e r i a l counts were found i n the control prawns followed by the NMAP and the CMAP prawns respectively (Figure 17). The i n i t i a l counts of t o t a l aerobic and anaerobic psychrotrophic bacteria were about l o g1 0 5.7. There was no si g n i f i c a n t difference (P>0.05) between t o t a l aerobic psychrotrophic b a c t e r i a l count (TSA) and t o t a l anaerobic psychrotrophic b a c t e r i a l count (TSN) i n prawns 6.50' 0 3 6 9 12 15 18 Days of Storage Figure 16. T r i a l 1: Inner tissue pHs of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 72 Figure 17. T r i a l 1: Total aerobic and t o t a l anaerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C (+ = aerobic, - = anaerobic) 73 from a l l treatments during storage. TSA and TSN of the control prawns increased most rapidly and reached about l o g1 0 8.5 within 6 days of storage. TSA and TSN of the NMAP prawns increased less rapidly than those i n the control prawns and gradually l e v e l l e d o f f toward the end of storage. In the CMAP prawns, however, TSA and TSN remained close to the i n i t i a l l e v e l u n t i l day 3 before they increased gradually at a slower rate than those i n the NMAP prawns u n t i l the end of storage. Changes i n TSA and TSN during storage of each treatment were s i g n i f i c a n t (P<0.01, Table 6). Significant differences were also found among treatments for both TSA and TSN, on every sampling day (Table 7). 4.1.5. K-values Similar K-values were found i n a l l samples up to day 6 of storage (Figure 18). After day 6 to the end of the storage, the NMAP prawns had higher K-values than the CMAP prawns. Changes i n K-value i n each treat-ment during 6 days storage as well as 18 days storage were s i g n i f i c a n t (P<0.01, Table 6). The difference among K-values of the CMAP and NMAP prawns was si g n i f i c a n t only on day 9 (P<0.05, Table 7). No ATP was detected i n the muscle tissue of the raw material prawns. ADP concentration decreased most rapidly i n the CMAP prawns followed by the control and the NMAP prawns respectively up to approximately day 12 (Figure 19). After that, the ADP concentration i n the CMAP prawns remained about the same while ADP concentration i n the NMAP prawns continued to decrease. Only the changes i n the ADP concentration of the CMAP prawns were s i g n i f i c a n t during 6 days storage (P<0.05, Table 6). During 18 days storage, ADP concentration changed s i g n i f i c a n t l y i n both the CMAP and the NMAP prawns (P<0.01, Table 6). Differences among the 74 Figure 18 T r i a l 1: K-values of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 75 8 1' _ 0 3 6 9 12 15 18 Days of Storage Figure 19. T r i a l 1: Adenosine diphosphate concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 76 ADP concentrations of the prawns from these three treatments were si g n i f i c a n t (P<0.01, Table 6) only on day 3. AMP concentrations i n the control, the CMAP, and the NMAP prawns de-creased very rapidly i n a similar manner (Figure 20). After day 6, the decrease i n the AMP concentration i n the CMAP and the NMAP prawns slowed down u n t i l the end of storage. In each treatment, AMP concentration changed s i g n i f i c a n t l y (P<0.01) during both storage periods (Table 6). But there were no s i g n i f i c a n t differences (P>0.05) among the AMP concentrations of prawns from these treatments on each sampling day (Table 7) throughout the storage. IMP concentration decreased rapidly i n a l l samples with the most rapid decrease occurring i n the control prawns followed by the NMAP and the CMAP prawns respectively (Figure 21). Changes i n the IMP concentra-tio n i n the control and the NMAP prawns during 6 days storage were si g n i f i c a n t (P<0.01, Table 6). A s i g n i f i c a n t change i n the IMP concentration of the CMAP prawns was found during 18 days storage (Table 6) . There were no s i g n i f i c a n t differences among the IMP concentrations of these prawns on each sampling day during storage except on day 9 (Table 7). Inosine concentration decreased most rapidly i n the control prawns (Figure 22). In the NMAP prawns, inosine increased and then decreased very rapidly. Inosine i n the CMAP prawns also increased, but more slowly and for a longer time compared to that i n the NMAP prawns, and then decreased at a slower rate than i n the NMAP prawns. Signifi c a n t changes in the inosine concentration were found only i n the NMAP prawns during 18 days storage (P<0.01, Table 6). Inosine concentrations of these (CMAP 77 F i g u r e 20. T r i a l 1: A d e n o s i n e monophosphate c o n c e n t r a t i o n s o f prawns s t o r e d unde r a e r o b i c c o n t r o l , c a r b o n d i o x i d e , and n i t r o g e n a tmosphe re s a t 1°C 7 8 60 Days of Storage Figure 21. Trial 1: Inosine monophosphate concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 79 Figure 22. T r i a l 1: Inosine concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 80 and NMAP) prawns were s i g n i f i c a n t l y different only on day 9 and day 18 (Table 7). Hypoxanthine developed gradually at a similar rate i n prawns from a l l treatments up to day 6 (Figure 23). After the f i r s t 6 days of stor-age, hypoxanthine concentration increased very rapidly i n the NMAP prawns u n t i l day 9 after which increases i n hypoxanthine concentration occurred slowly. After day 6, hypoxanthine concentration increased at a slower rate i n the CMAP prawns than i n the NMAP prawns. These changes i n hypoxanthine concentration i n each treatment were s i g n i f i c a n t during 6 days storage as well as 18 days storage (Table 6). On day 12 and day 18, there were s i g n i f i c a n t differences (P<0.01) among the hypoxanthine concentrations i n the CMAP and the NMAP prawns (Table 7). 4.1.6. Trimethylamine-nitrogen concentration TMAN concentration increased very slowly i n the prawns from each treatment up to day 6 (Figure 24). In the NMAP prawns, TMAN concentra-tio n then increased rapidly u n t i l the l a s t day of storage. TMAN concentration i n the CMAP prawns slowly increased up to day 9 and then increased rapidly at a rate similar to that observed for the NMAP prawns u n t i l the l a s t day of storage. The NMAP prawns had higher concentrations of TMAN than the CMAP prawns at the end of T r i a l 1. Changes i n the TMAN concentration were not s i g n i f i c a n t i n the CMAP prawns but s i g n i f i c a n t i n the control and the NMAP prawns during the f i r s t 6 days of storage (P<0.01, Table 6). During 18 days storage, TMAN concentration i n the CMAP as well as the NMAP prawns changed s i g n i f i c a n t l y (P<0.01, Table 7). Significant differences (P<0.01) among the TMAN concentrations of these (CMAP and NMAP) treatments were found (P<0.01, Table 6) from day 9 81 aFirSo\rL2contro\la^«bo^S"n:„d0nC:ntrati<,nS °f ^ S t°™* , caroon dioxide, and nitrogen atmospheres at 1 C 82 Figure 24. T r i a l 1: Trimethylamine-nitrogen concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 83 onwards. TMAN concentrations of the NMAP prawns were s i g n i f i c a n t l y higher (P<0.05) than those of the control and the CMAP prawns. 4.1.7. Sensory evaluation Changes i n the sensory characteristics among treatments were not evenly developed. Samples having an overall sensory score of less then 5.00 were considered to be of an unacceptable q u a l i t y . At the end of the storage time, the overall sensory scores of above 5.00 indicated that a l l prawns except control samples were s t i l l acceptable from a sensory point of view (Figure 25). However, as shown i n Figure 26, the NMAP prawns could have been rejected on day 9 of storage based on the scores of raw prawn meat odour which i s one of the prime freshness c r i t e r i a often used by customers. Furthermore, a l l samples might have also been rejected based on the scores of cooked meat odour as shown i n Figure 27. There was no clear trend of either the NMAP or the CMAP being more capable of maintaining prawn sensory characteristics except for colour and texture of cooked meat, where the NMAP prawns generally had better scores than the CMAP prawns (Figures 28 and 29). Scores for raw prawn meat colour and scores for cooked prawn meat flavour were as shown i n Figures 30 and 31 respectively. The results from the Friedman two-way ANOVA on these sensory data (Table 8) showed that the panel did not detect any s i g n i f i c a n t changes i n prawns from each treatment with storage l i f e up to day 6 except for the cooked prawn meat colour of the NMAP prawns. The panel also did not detect any s i g n i f i c a n t changes i n sensory characteristics i n prawns from each treatment with storage l i f e up to day 18 except for the raw prawn meat odour and the cooked prawn meat odour of the NMAP prawns. The 84 O O if) o w c Q) Ul > o B Control — • — CMAP A NMAP 12 15 18 Days of Storage Figure 25. T r i a l 1: Overall sensory scores of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 85 (D i_ o o 00 i_ D O "O O •4-> O C CL o cn B Control — • — CMAP — ± — NMAP 12 15 18 Days of Storage Figure 26. T r i a l 1: Scores for raw prawn meat odour of prawns stored under aerobic c o n t r o l , carbon dioxide, and nitrogen atmospheres at 1°C 86 Figure 27. T r i a l 1: Scores for cooked prawn meat odour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 87 i_ O O 00 D o o o a (D 2 CL T> (D o o o B Control — • — CM/AP — • — NMAP 9 12 15 18 Days of Storage Figure 28. T r i a l 1: Scores for cooked prawn meat colour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 88 Figure 29. T r i a l 1: Scores for cooked prawn meat texture of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at X c 89 Figure 30 T r i a l 1: Scores for raw prawn meat colour of prawns stored under aerobrc control, carbon dioxide, and nitrogen atmospheres at 1°C 90 at 91 Table 8. F - s t a t i s t i c s ( a ) from Tukey's test on data of T r i a l 2 (data of 12 day) A l l Indicators(b) Control CMAP NMAP Treatments Exudate NV (c) NV NV NS (d) PH 39. .712 ** 24. .516 ** 15. .838 ** 19. .615 ** TSA 88. .706 ** 15. .673 ** 56. .071 ** 3. .570 * TSN 87. .449 ** 16. .708 ** 49, .750 ** NS SPA 301. .252 ** 72. .660 ** 199. .290 ** NS SPN 318. .807 ** 26. .914 ** 218. .110 ** NS TMAN 187. .932 ** 5. .557 * 31. .613 ** NS TVBN 17. .966 ** 12. .693 ** 16. .430 ** 3. .625 * WSP 1065. .782 ** 9. .946 ** 31. .773 ** 4. .136 * SSP 14. .386 ** 11. .547 ** 8. .324 ** 8. .188 ** L 4. .659 * 8, .179 ** 12. .728 ** 4. .867 * a NS NS 16. .413 ** 14. .274 ** b NS NS NS NS RC NV NV NV NS RO NV NV NV NS CC NV NV NV 10. .414 ** CO NV NV NV NS CF NV NV NV NS CT NV NV NV NS Overall NV NV NV NS (a) * = s i g n i f i c a n t at 5% le v e l ** = s i g n i f i c a n t at 1% le v e l (b) See abbreviation l i s t (c) One or more of groups has no variance (d) No significance 92 panel, however, did detect differences i n the cooked prawn meat colour, the raw prawn meat odour, and the raw prawn meat colour among prawns of different treatments during the f i r s t 12 days of storage. In the two-way analysis of variance on each sensory c h a r a c t e r i s t i c , s i g n i f i c a n t differences among judges were evident i n every sensory characteristic (P<0.01). 4.1.8. Hunter L, a, b values While the Hunter L value of the control prawns remained the same during the f i r s t 6 day of storage, i t increased i n both the CMAP and the NMAP prawns i n a similar manner with the CMAP having a s l i g h t l y higher value by the end of 18 days of storage (Figure 32). In each treatment, the Hunter L value changed s i g n i f i c a n t l y (P<0.01) i n the CMAP and the NMAP but not the control prawns during 6 days storage (Table 6). During 18 days storage, the Hunter L value changed s i g n i f i c a n t l y (P<0.01) i n both the CMAP and the NMAP prawns (Table 6). On day 3 and day 6 of the storage, the Hunter L values among a l l three treatments were s i g n i f i c a n t l y different (P<0.05, Table 7). After that,, there were no sig n i f i c a n t differences i n the Hunter L value (between the CMAP and the NMAP prawns). While the Hunter a value of the control prawns changed very l i t t l e , i t increased i n both the CMAP and the NMAP prawns (Figure 33) . The Hunter a value of the CMAP prawns increased u n t i l day 6 and then just s l i g h t l y increased u n t i l the end of storage. The Hunter a value of the NMAP prawns dramatically increased u n t i l day 9 and then remained about the same u n t i l the end of storage. During 6 days storage, changes i n the Hunter a value were s i g n i f i c a n t i n the CMAP prawns (P<0.01, Table 6). At 42 37' 0 3 6 9 12 15 18 Days of Storage Figure 32. T r i a l 1: Hunter L values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 94 0 3 6 9 12 15 18 Doys of Storage Figure 33. T r i a l 1: Hunter a values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 95 12.5 10.0' :  0 3 6 9 12 15 18 Days of Storage Figure 34. T r i a l 1: Hunter b values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 96 the same storage period, however, changes i n the Hunter a value i n the NMAP prawns were marginally s i g n i f i c a n t (P=0.058). The Hunter a values of the CMAP and the NMAP prawns changed s i g n i f i c a n t l y (P<0.01) during 18 days storage. When comparing the Hunter a values among treatments, s i g n i f i c a n t differences were found on day 6, day 12, and day 15 (Table 7). The Hunter b value increased i n a l l prawn samples evaluated (Figure 34). The NMAP prawns had s l i g h t l y higher Hunter b values than the CMAP prawns during most of the storage t r i a l . The Hunter b value i n the NMAP prawns increased u n t i l day 9 and then slowly decreased u n t i l the end of storage. During 6 days storage of each treatment, the Hunter b value of prawns changed s i g n i f i c a n t l y (Table 6). Significant changes of the Hunter b values i n the CMAP and the NMAP prawns were also observed during 18 days storage. But there was no si g n i f i c a n t difference i n the Hunter b value among treatments on any sampling day throughout the storage. 4.2. Trial 2 In T r i a l 2 of this study, sampling was carried out every 4 days up to day 28 for a l l treatments (the control, the CMAP, and the NMAP prawns). 4.2.1. Headspace gas composition Changes i n the headspace gas composition i n the CMAP and the NMAP bags are i l l u s t r a t e d i n Figures 35 and 36 respectively. A s l i g h t i n-crease i n carbon dioxide concentration gradually took place i n the NMAP bags, similar to that observed i n T r i a l 1. Changes i n nitrogen concentration i n the bags were proportional to the changes i n the carbon 97 DayO Day8 Day16 Day28 Figure 35. T r i a l 2: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the CMAP bags stored at 1°C 98 C o CL 100 80 60 40 20 0 0.9^6 91.04 93.21 8.86 MM 6.67 86.54 12.95 i.5' DayO Day8 Day16 Day28 Figure 36. T r i a l 2: Concentrations of carbon dioxide, oxygen, and nitrogen gas i n the NMAP bags stored at 1°C 99 dioxide concentration. Carbon dioxide concentration i n the CMAP bags dropped between day 0 and 4 and l a t e r slowly increased. Carbon dioxide concentrations i n the CMAP bags during storage d i f -fered s i g n i f i c a n t l y (F=17.472, P<0.01) with that of day 0 being the highest and s i g n i f i c a n t l y different from the re s t . A s i g n i f i c a n t d i f -ference i n carbon dioxide concentration i n the NMAP bags during storage was also found (F=8.922, P<0.01). 4.2.2. Tissue pH Changes i n the tissue pH of prawns are shown i n Figure 37. The prawns, at the time of packing, had a pH of about 7.5. After a s l i g h t i n i t i a l drop, the pH of the control prawns increased over storage time. The pH of the NMAP prawns remained i n the range of 7.3 to 7.5 over the 28 days storage period. pH of the CMAP prawns dropped to 6.5 within 4 days of storage. The CMAP prawns increased s l i g h t l y to pH 6.7 by day 12 where they remained for the duration of the storage t r a i l . During 12 days storage, there was a si g n i f i c a n t change (P<0.01) i n prawn tissue pH i n each treatment (Table 9). Differences i n tissue pH among treatments were si g n i f i c a n t on every sampling day during 12 days storage (P<0.01, Table 9). Tissue pH values of the CMAP prawns were always s i g n i f i c a n t l y lower (P<0.05) than those of the NMAP prawns which were also always s i g n i f i c a n t l y lower (P<0.05) than those of the control prawns. 4.2.3. Exudate formation Changes i n exudate formation i n prawns are shown i n Figure 38. Rise and f a l l of exudate values was found i n this t r i a l . This was probably the result of the preparation procedure used to remove the roe from the 100 Figure 37. T r i a l 2: Inner tissue pHs of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C Table 9. F-statistics (a) from ANOVA on data 12 days storage of Tri a l 2 Variables (b) Control CMAP NMAP Exudate (c) 3. .847 * 0. 318 2 .507 pH 39. .712 ** 24. 516 ** 15 .838 ** TSA 88. .706 ** 15. 673 ** 56 .071 ** TSN 88. .449 ** 16. 708 ** 49 .750 ** SPA 301. .252 ** 72. 660 * 199 .290 ** SPN 318. .807 ** 26. 914 ** 218 .110 ** TMAN 184. .932 ** 5. 557 * 31 .613 ** TVBN 17. .966 ** 12. 693 ** 16 .430 ** WSP 1065. .782 ** 9. 946 ** 31 .773 ** SSP 14. .386 ** 11. 547 ** 8 .324 ** L 4. .659 ** 8. 179 ** 12 .728 ** a 1. .083 4. 040 16 .413 ** b 3. .059 3. 504 0 .316 Variables Day4 Day8 Dayl2 Exudate 0. .750 12. .761 ** 0. .727 pH 283 .978 ** 146. .184 ** 48. .533 ** TSA 31. .903 ** 26. .760 ** 95. .573 ** TSN 32 .411 ** 19. .598 ** 158. .447 ** SPA 38 .661 ** 712. .262 ** 115. .076 ** SPN 12. .226 ** 37. .081 ** 60. .046 ** TMAN 2 .448 10. .265 * 10. .822 * TVBN 4. .732 5. .463 * 15. .655 ** WSP 15, .322 ** 50. .944 ** 174. .210 ** SSP 8, .439 * 8. .037 * 38. .249 ** L 0. .300 11. .838 ** 41. .885 .JUJL. a 4, .579 10. .633 * 48. .473 ** b 0. .204 6. .588 * 2. .676 (a) * = significant at 5% level ** = significant at 1% level (b) See Abbreviation List (c) Only data of day 4 to day 12; no exudate on day 0 102 16 Days of Storage Figure 38. T r i a l 2: Exudate volumes of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 103 prawns. Roe were removed by opening up the carapaces at the abdominal area, and scraping them off with a spatula. This procedure, although performed c a r e f u l l y , damaged the carapaces and exposed the abdominal tissue of the prawn. Generally, more exudate was observed i n the CMAP prawns than i n the NMAP or control prawns. A s i g n i f i c a n t change (P<0.05) i n exudate volume was found i n the control prawns during 12 days storage (Table 9). Only on day 8, differences i n exudate volume among these treaments were s i g n i f i c a n t (P<0.01, Table 9). 4.2.4. Microbiology Figures 39 to 40 show the changes i n TSA and TSN of the control, the CMAP, and the NMAP prawns respectively. The highest TSA and TSN were found i n the control prawns followed by the NMAP and the CMAP prawns respectively. On the whole, aerobic psychrotrophic bacteria grew more slowly i n the NMAP and CMAP prawns with slowest growth observed i n the CMAP prawns. Changes i n SPA and SPN of the control, the CMAP, and the NMAP prawns were shown i n Figures 41 to 42 respectively. The raw material prawns (day 0) had a s i g n i f i c a n t l y higher population of sulphide-producing aerobes than sulphide-producing anaerobes (T=4.770, P=0.041). By the end of 28 days storage, the aerobic and anaerobic populations of sulphide-producing psychrotrophic bacteria were similar i n a l l treatments. Aerobic sulphide-producing psychrotrophic bacteria grew at a simi l a r rate i n the control and the NMAP prawns, with the population l e v e l l i n g off to about 105 cfu/gm by day 8. In the CMAP prawns, aerobic sulphide-producing psychrotrophic bacteria grew at a slower rate and l e v e l l e d off slowly by the end of storage. Anaerobic sulphide-producing psychrotrophic 104 Figure 39. T r i a l 2: Total aerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 105 Figure 40. T r i a l 2: Total anaerobic psychrotrophic b a c t e r i a l counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 106 Figure 41. T r i a l 2: Total aerobic sulphide-producing psychrotrophic bacterial counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 107 Figure 42. T r i a l 2: Total anaerobic sulphide-producing psychrotrophic bacterial counts of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 108 bacteria grew more rapidly than aerobic sulphide-producing psychrotrophic bacteria during the f i r s t 12 days of storage i n the control and the NMAP prawns. Anaerobic sulphide-producing psychrotrophic bacteria grew much less rapidly i n the CMAP prawns, but the population by day 28 was similar to that observed for the NMAP prawns. Changes i n TSA, TSN, SPA, and SPN during 12 day storage of each treatment were s i g n i f i c a n t (P<0.01, Table 9). Significant differences i n TSA, as well as TSN, SPA, and SPN among treatments were also observed on each sampling day during 12 days storage (P<0.01, TAble 9). 4.2.5. Trimethylamine-nitrogen concentration Changes i n TMAN concentrations are shown i n Figure 43. The TMAN concentration increased very slowly i n the control and the NMAP prawns up to day 4, and i n the CMAP prawns up to day 8. TMAN concentration i n the NMAP prawns then rapidly increased u n t i l the l a s t day of storage. After day 8, the TMAN concentration i n the CMAP prawns rapidly increased u n t i l the end of storage. The TMAN concentrations i n the control prawns up to day 16 were lower than those i n the NMAP prawns but higher than those i n the CMAP prawns. After day 16, the TMAN concentration i n the control prawns suddenly decreased before starting to increase again u n t i l the end of storage. At the end of storage, the highest concentration of TMAN was found i n the NMAP prawns followed by the CMAP and the control prawns respectively. Changes i n TMAN concentration i n these prawns during 12 storage were si g n i f i c a n t i n each treatment (Table 9). While no s i g n i f i c a n t difference i n TMAN concentration among treatments was observed on day 0, s i g n i f i c a n t differences were found on day 8 and day 12 (Table 9). 109 90 0 8 16 24 4 12 20 28 Days of Storage Figure 43. T r i a l 2: Trimethylamine-nitrogen concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 110 4.2.6. Total v o l a t i l e basic nitrogen concentration The TVBN concentration i n a l l prawns (Figure 44) increased s l i g h t l y at a similar rate up to day 4. After day 4, i t increased rapidly at a simil a r rate i n the control and the NMAP prawns. After day 20, the TVBN concentration i n the NMAP prawns dropped dramatically while the TVBN concentration i n the control prawns continued to increase, but at a much slower rate than before. The TVBN concentration i n the CMAP prawns increased gradually u n t i l day 16, and then remained at about 100 mgN/lOOgm tissue u n t i l the end of storage. Changes i n TVBN concentration i n each treatment during 12 days storage were s i g n i f i c a n t (P<0.01, Table 9). But s i g n i f i c a n t differences i n TVBN concentration among treatments were observed only on day 8 and day 12 (Table 9). TVBN concentrations of the CMAP prawns were s i g n i f i c a n t l y (P<0.05) lower than those of the NMAP and the control prawns during 12 days storage. 4.2.7. Soluble proteins Changes i n the concentrations of WSP and SSP of the control, the CMAP, and the NMAP prawns are shown i n Figures 45 to 46. The concentration of SSP was higher than that of WSP at the beginning of storage. Generally, SSP concentration increased at f i r s t , then gradually decreased with the storage time i n a l l prawns while the WSP concentration increased gradually and then decreased continuously u n t i l the end of storage. The highest concentration of the WSP was i n the control prawns followed by the NMAP and the CMAP prawns respectively. In contrast to the development of the WSP, the highest concentration of the SSP was i n the CMAP prawns followed by the NMAP and the control prawns respectively. I l l Figure 44. T r i a l 2: Total v o l a t i l e basic nitrogen concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 112 Figure 45. T r i a l 2: Water-soluble protein concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 113 Figure 46. T r i a l 2: Salt-soluble protein concentrations of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 114 During the l a s t week of storage of the control prawns, however, there was a sudden increase i n the SSP concentration which, after day 24, immediately decreased u n t i l the end of storage. Changes i n WSP and SSP for prawns i n each treatment group were si g n i f i c a n t during 12 days storage (P<0.01, TAble 9). WSP as well as SSP of these treatments were s i g n i f i c a n t l y different on each sampling day during 12 days storage (Table 9). 4.2.8. Sensory evaluation By using an overall sensory score of more than or equal to 5.00 as an indication of the acceptable quality of the prawns, the scores i n Figure 47 indicated that the panel rejected the control prawns on day 8 and the CMAP and the NMAP prawns on day 12. Scores for each sensory characteristic (RC, RO, CC, CO, CF, and CT) were as shown i n Figures 48 to 53 respectively. For the CMAP prawns at day 12, however, the average scores for the raw prawn meat odour and colour and the cooked prawn meat colour were s t i l l i n the acceptable range. On day 8, while the average score for raw prawn meat odour of the CMAP prawns was s t i l l acceptable, the scores for the control and the NMAP prawns were i n the rejection range (Figure 48). Raw prawn meat colour of the NMAP prawns was s t i l l acceptable by day 12 while raw prawn meat colour of the control and the CMAP prawns were considered unacceptable (Figure 49). Cooked prawn meat odour of a l l samples were rejected by day 4 (Figure 50). On day 8, while the cooked prawn meat colour of the NMAP and the control were s t i l l quite acceptable, the cooked prawn meat colour of the CMAP prawns were rejected (Figure 51). On day 8, the cooked prawn meat flavour of the CMAP and the NMAP prawns were s t i l l acceptable but that of the control prawns was not 115 Figure 47. T r i a l 2: Overall sensory scores of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 116 Figure 48. T r i a l 2: Scores for raw prawn meat colour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 117 Figure 49. T r i a l 2: Scores for raw prawn meat odour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 118 Figure 50. T r i a l 2: Scores for cooked prawn meat colour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 119 Figure 51. T r i a l 2: Scores for cooked prawn meat odour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 120 Figure 52. T r i a l 2: Scores for cooked prawn meat flavour of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 121 <D i_ O O Ul fi? D X O C CL "O 0) _x" o o o 8 7 6 5 4 3 2 0 8 12 Days of Storage FJ£?« T r i a l u2 : S c ° r e S f o r c o o k e d P « ~ meat texture of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at Table 10. F - s t a t i s t i c s (a) from Friedman two-way ANOVA on sensory data of T r i a l 2 I. Data of 12 days storage Variables (b) Control CMAP NMAP RC 6.600 10.350 * 4.500 RO 12.250 ** 3.350 8.150 * CC 10.350 ** 4.500 2.600 CO 10.100 * 3.240 6.150 CF 11.450 ** 4.020 6.180 CT 1.020 5.940 3.000 I I . Data of individual sampling day Variables Day4 Day8 Dayl 2 RC 1.750 1.583 1.900 RO 1.583 2.083 7.000 * CC 4.083 5.583 2.083 CO 0.083 1.900 1.083 CF 0.583 0.900 6.583 * CT 0.083 5.375 1.583 (a) * = s i g n i f i c a n t at 5% le v e l ** = s i g n i f i c a n t at 1% leve l (b) See Abbreviation L i s t 123 (Figure 52). By day 8, cooked prawn meat texture score for the CMAP prawns f e l l into the rejection range but those of the control and the NMAP prawns were s t i l l i n the acceptable range (Figure 53). The results from Friedman two-way ANOVA on these sensory data (Table 10) showed that the panel detected s i g n i f i c a n t changes i n : raw prawn meat odour, cooked prawn meat colour, odour, and flavour of the control prawns; raw prawn meat colour of the CMAP prawns; and raw prawn meat odour of the NMAP prawns. The panel detected only the s i g n i f i c a n t (P<0.05) differences i n raw prawn meat odour and i n cooked prawn meat flavour of prawns among treatments on day 12 of storage. In the two-way analysis of variance on each sensory c h a r a c t e r i s t i c , s i g n i f i c a n t differences among judges were evident i n every sensory char a c t e r i s t i c (P<0.01). 4.2.9. Hunter L, a, b values The Hunter L values (Figure 54) of a l l prawns were s i m i l a r up to day 4. Then the Hunter L value of the control prawns started to decrease gradually, while those of the CMAP prawns continued to increase consistently and those of the NMAP prawns increased only s l i g h t l y . Changes i n the Hunter L value of each treatment were s i g n i f i c a n t (P<0.01, Table 9). But si g n i f i c a n t differences (P<0.01) i n the Hunter L value among treatments were observed on day 8 and day 12. The Hunter a value (Figure 55) was highest i n the NMAP prawns f o l -lowed by the CMAP and the control prawns respectively. The Hunter a values of the NMAP and the CMAP prawns increased but the Hunter a values of the control prawns decreased during storage. During 12 days storage, changes i n the Hunter a. value were si g n i f i c a n t only i n the NMAP prawns 124 Figure 54. T r i a l 2: Hunter L values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 125 Figure 55. T r i a l 2: Hunter a values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 126 Figure 56. T r i a l 2: Hunter b values of prawns stored under aerobic control, carbon dioxide, and nitrogen atmospheres at 1°C 127 (P<0.01, Table 9). Significant differences i n the Hunter a value among treatments were found on day 8 and day 12. The Hunter b values (Figure 56) of the control and the CMAP prawns changed s l i g h t l y during the f i r s t 4 days of storage and then increased u n t i l day 8. After that, the Hunter b values of the control prawns dropped dramatically while those of the CMAP prawns continued to increase. The Hunter b values of the NMAP prawns increased gradually u n t i l day 4 and decreased gradually after day 4 back to the i n i t i a l value by day 12. No s i g n i f i c a n t changes (P>0.05) i n the Hunter b value were found among treatments during storage. A s i g n i f i c a n t difference i n the Hunter b value among treatments was observed on day 8 only. 4.2.10. Factor analysis In the results from factor analysis, v a r i a t i o n i n the multivariate data was summarized by 4 factors or p r i n c i p a l components. The f i r s t extracted factor accounted for 59.07% of the data variance. The f i r s t two extracted factors accounted for 80.08% of the data variance. The f i r s t three extracted factors accounted for 90.90% of the data variance. A l l four extracted factors accounted for 100.00% of the data variance. The sorted rotated factor loadings (Table 11) shows that Factor 1, a broad factor, contained a l l microbiological variables, odour score and colour score of raw prawn meat, TMAN concentration, odour score and flavour score of cooked prawn meat, WSP, and over a l l sensory score. Factor 2 described colour: Hunter L and a values and SSP. Factor 3 was a measure of colour and texture of cooked prawn meat, exudate volume, and pH. Factor 4 was a measure of Hunter b value and TVBN concentration. Table 11. Sorted rotated factor loadings from factor analysis of data( a ) Variables( b ) FACT0R1 FACT0R2 FACT0R3 FACT0R4 TSA 1. 000 0. .000 0 .000 0. .000 TSN 0. 980 0. .000 0 .000 0. ,000 RO -0. 950 0. .000 0 .000 0. .000 SPA 0. 946 0. .000 0 .000 0. .000 SPN 0. 908 0. .000 0 .000 0. .000 TMAN 0. 862 0. .290 0 .000 0. .000 CF -0. 857 0. .000 0 .270 0. ,000 WSP 0. 833 -0. .397 0 .000 0. .000 OVERALL -0. 830 0. .000 0 .342 0. .000 CO -0. 823 0. .000 0 .306 0. .000 RC -0. 765 0. .000 0 .000 -0. .581 a 0. 000 0 .933 0 .000 0. .000 SSP 0. 000 0. .881 0 .000 0. .000 L 0. 000 0. .858 0 .000 0. .325 CC 0. 000 0 .327 0 .825 -0. .272 DRIP 0. 000 0, .000 -0 .822 0. .000 pH 0. 651 -0. .378 0 .691 0. .000 CT -0. 311 0, .000 0 .626 0. .319 b 0. 000 0. .000 0 .000 0. .801 TVBN 0. 505 0. .000 0 .000 -0. .634 The above factor loading matrix has been rearranged so that the columns appear in decreasing order of variance explained by factors. The rows have been rearranged so that for each successive factor, loadings greater than 0.5000 appear f i r s t . Loadings less than 0.2500 have been replaced by zero. See Abbreviation List Table 12. Rotated factor loadings from factor analysis of data Variables( a ) FACT0R1 FACT0R2 FACT0R3 FACT0R4 EXUDATE 0. .099 0. .186 -0 .822 -0. .012 PH 0. .651 -0. .378 0 .691 -0. .035 TSA 1. .000 0. .050 0. .085 0. .045 TSN 0. .980 0, .066 0 .019 0. .071 SPA 0. .946 0. .109 -0 .043 0. .102 SPN 0. .908 0. .158 -0. .121 -0. .065 TMAN 0. .862 0. .290 0 .138 -0. .210 TVBN 0. .505 -0. .013 -0. .128 -0. .634 WSP 0. .833 -0. .397 -0. .024 -0. .108 SSP -0. .022 0. .881 -0, .090 -0. .157 L 0. .048 0. .858 -o . .105 0. .325 a 0. .096 0. .933 0. .073 0. .025 b 0. .190 0. .137 -0. .174 0. .801 OVERALL -0. .830 0. .099 0. .342 -0. .114 RC -0. .765 0. .033 -0. .091 -0. .581 RO -0. .950 0. .051 0. .006 0. .022 CC -0. .197 0. .327 0. .825 -0. .272 CO -0 . ,823 0. .000 0. .306 0. .019 CF -0. .857 0. .179 0. .270 -0. .042 CT -0. .311 -0. .196 0. .626 0. .319 See Abbreviation L i s t 130 1.0 0.5 H Hunter a S S H u n t e r L CM ° o.o -J O a Li_ RO CF Overall CC TMAN Exudate Hunter b SPN TVBN CT -0.5 pH w s p 1.0 1 ' I I I I I I 1 | I 1 1 1 1 I 1 1 1 1 •1.0 -0.5 0.0 0.5 1.0 F a c t o r 1 Figure 57. T r i a l 2: Plots of Factor 1 against Factor 2 of rotated factor loadings from the factor analysis on data of 12 days storage of T r i a l 2 131 The rotated factor loadings from factor analysis of the data was shown i n Table 12. The plot of the rotated factor loadings for Factor 1 versus Factor 2 i s shown i n Figure 57 and indicates clustering of several Groups of variables: odour of raw and cooked prawn meat, colour of raw and flavour of cooked prawn meat, and overall sensory scores; SSP concentration, and Hunter L and a values; exudate volume and Hunter b value; WSP concentration and trimethylamine-nitrogen concentration; and a l l microbiological data. 4.2.11. Stepwise discriminant analysis Stepwise discriminant analysis on the "multivariate data" with TMAN concentration as the grouping variable resulted i n 9 variables. These variables were SPA, pH, TVBN, CO, SSP, CF, WSP, CT, and Hunter L value ranked i n descending order of U - s t a t i s t i c . The smaller the U - s t a t i s t i c becomes, the closer i s the c l a s s i f i c a t i o n coming to perfection (Powers and Ware, 1986). C l a s s i f i c a t i o n matrix and Jackknife c l a s s i f i c a t i o n obtained from this stepwise discriminant analysis were 100% and 94.4% correct. When considering the p r a c t i c a l i t y , using 9 tests to evaluate prawn quality i s too time-consuming, laborious, and co s t l y . To overcome this s i t u a t i o n , a minimum number of test must be selected. In a case where one test i s highly correlated with another or moderately correlated with several and these other tests have already been included i n the process, the correlated terms would not be providing new information useful for discrimination but only information p a r t i a l l y redundant with that already available (Powers and Ware, 1986). They commented that a variable which by i t s e l f was not a great discriminator but which was not well correlated 132 with any other variable already i n use was more l i k e l y to add to the discrimination power than one which was correlated. Therefore, more stepwise discriminant analyses were carried out on many combinations of variables. The combination of SPA and pH produced the highest (97.2%) success i n both c l a s s i f i c a t i o n matrix and Jackknife c l a s s i f i c a t i o n . In fa c t , the result from the Jackknife c l a s s i f i c a t i o n was better than when a l l variables (tests) were used i n the analysis. As a result of the stepwise discriminant analysis on pH and SPA data using the TMAN concentration as the grouping variable, the coefficients for 2 canonical variables were produced as shown i n the following equations: Canonical variable 1 = 3.00114 [pH] - 1.99857 [SPA] - 14.06103 Canonical variable 2 = 2.45639 [pH] - 0.06084 [SPA] - 18.10119 The coefficients of variables describe the two canonical variables that are used for v i s u a l discrimination of the group i n two-dimensional space. The plot was as i l l u s t r a t e d i n Figure 59. The result produced only 1 incorrect c l a s s i f i c a t i o n (out of 36 cases) which was indicated by the c i r c l e on the p l o t . This plot belonged to the data of the second bag of the CMAP prawns of day 8. 133 3.0 CM 2.0 -CD < i-o < > o.o _ l < O < i.o — -2.0 -3.0 c c c cc c c BP 1 I I I I i — r ~ i — i — r ~ i — r ~ 6.0 -4.0 -2 .0 0.0 I I I I I I I I I 2.0 4.0 6.0 CANONICAL VARIABLE 1 Figure 58. T r a i l 2: Canonical plots of prawn samples c l a s s i f i e d prawns by stepwise discriminant analysis of pH and SPA A = Grade I: < 1.00 mg TMAN/ 100 gm tissue B = Grade I I : 1.01 to 5.00 mg TMAN/ 100 gm tissue C = Grade I I I : > 5.00 mg TMAN/ 100 gm tissue 134 5. DISCUSSION 135 5. DISCUSSION 5.1. Headspace gas compositions in the bags The results from the gas chromatographic analysis showed an immedi-ate decrease in the concentration of carbon dioxide in the CMAP bags. This reduction of the carbon dioxide concentration in the CMAP bags was mainly the result of a portion of carbon dioxide gas dissolving into the liquid phase of prawn tissue. Compared to most other gases, carbon dioxide is more soluble in water. Its solubility increases as temperature decreases. Gases transmission through the film would be negligibly small since the film is practically gas-impermeable. During storage of the prawns, the carbon dioxide concentration in the headspace atmosphere increased due to microbial respiration or biochemical reactions. Collapse or deformation of the CMAP bags occurred in this study as a result of carbon dioxide dissolving in the prawn muscle tissue. On the contrary, there was no such dramatic changes in the headspace gas composition in the NMAP bags. The concentration of carbon dioxide in the NMAP bags slightly increased as a result of microbial activities and tissue enzymatic reactions. The changes of the nitrogen concentrations in the NMAP bags were mainly proportional to the changes of carbon dioxide levels in the bags. 5.2. Tissue pHs The i n i t i a l pH of the pink prawns in this study was in agreement with the findings of Flick and Lovell (1972) and Bailey et al. , (1956). 136 The immediate drop i n the tissue pH of the CMAP samples happened at a time coinciding with the reduction of carbon dioxide concentrations i n the CMAP bags. This phenomenon was also reported i n a study by Lannelongue et al., (1982). An absence of the i n i t i a l pH drop was evident i n the control prawns of T r i a l 1 but not i n T r i a l 2. An i n i t i a l pH drop i n the NMAP prawns occurred i n both t r i a l s i n this study. This may be a result of glycolysis or the carbon dioxide dissolving into the prawn tissue or both. The tissue pHs of the NMAP and the control prawns increased as a result of microbial production of various spoilage compounds such as ammonia and amines. These results are i n accordance with the findings i n the changes i n the TVBN and the TMAN concentrations i n t h i s study. Since prawn tissue pHs were s i g n i f i c a n t l y different among treatments on every sampling day as well as during the whole storage period, i t might be a suitable index to help categorize prawns from the three treatments of the same storage period. The discriminating power of the tissue pH was evident i n the result of the discriminant analysis. 5.3. Exudate formation The greatest reduction i n tissue pH occurred i n the CMAP prawns which had the highest volume of exudate. This i s i n general agreement with several reports by Penny (1977), Khan (1977), Warriss (1982), and Warriss and Brown (1987) on porcine muscle. However, a low correlation between exudate volume and pH was evident (r=-0.492 i n T r a i l 1 and r=-0.562 i n T r i a l 2, Tables 12 and 14). Developments of the exudates i n the NMAP and the control prawns were i n accordance with the changes i n their tissue pHs. 137 Table 13. Correlation matrix from factor analysis on data of T r i a l 1 EXUDATE PH TSA TSN TMAN EXUDATE 1. 000 PH - o . 492 1. ,000 TSA 0. 265 0. 480 1. .000 TSN 0. 268 0. ,518 0, .985 1. .000 TMAN 0. 431 0. ,355 0. .587 0. .637 1.000 IMP -0 . 590 -0. 148 -0. .747 -0. .764 -0.783 ADP -0 . 638 0. 157 -0 .484 -0 .477 -0.603 AMP -0 . 700 0. 026 -0. .754 -0 .753 -0.647 HX 0. 497 0. 192 0. .620 0. .668 0.854 HXR - 0 . 223 -0. 502 -0. .565 -0. .604 -0.812 K 0. 652 0. 083 0. .721 0. .742 0.810 L 0. 485 0. 170 0. .605 0. .644 0.650 A 0. 500 0. 145 0. .527 0. .558 0.646 B 0. 584 -0. 054 0. .502 0, .507 0.513 RC 0. 032 0. 293 0. .291 0. .341 0.381 RO -0 . 187 -0. 397 -0. ,662 -0. .671 -0.632 CC -0 . 228 0. 643 0. ,390 0. .468 0.457 CO -0 . 604 0. 135 -0. .567 -0. .566 -0.521 CF -0 . 492 -0. 023 -0. .623 -0. .640 -0.594 CT -0 . 759 0. 484 -0. ,238 -0. .238 -0.423 OVERALL -0 . 613 0. 312 - -0. .394 -0. .364 -0.356 IMP ADP AMP HX HXR IMP 1. 000 ADP 0. 806 1. 000 AMP 0. 924 0. 817 1. ,000 HX -0 . 849 -0. 602 -0. ,782 1. .000 HXR 0. 754 0. 568 0. ,533 -0. .680 1.000 K -0 . 940 -0 . 733 -0. ,925 0. .926 -0.640 L -0 . 655 -0 . 387 -0. 624 0. .662 -0.472 A -0 . 659 -0. 417 -0. ,630 0. .648 -0.496 B -0 . 626 -0 . 521 -0. ,665 0. .565 -0.372 RC -0 . 323 -0 . 307 -0. 216 0. ,325 -0.446 RO 0. 658 0. 362 0. ,603 -0. .666 0.554 CC -0 . 272 -0 . 085 -0. ,169 0. .326 -0.480 CO 0. 773 0. 651 0. ,850 -0. .709 0.385 CF 0. 765 0. 637 0. 810 -0. .730 0.427 CT 0. 582 0. 636 0. ,662 -0. .533 0.171 OVERALL 0. 604 0. 523 0. 703 -0. .546 0.170 Continued next page 1 3 8 Table 13 continued.. K L A B RC K 1.000 L 0 .698 1.000 A 0 . 654 0 .707 1.000 B 0 . 622 0 .634 0 .785 1.000 RC 0 .279 0 .232 - 0 . 015 - 0 . 0 6 9 1 .000 RO - 0 . 6 6 6 - 0 . 566 - 0 . 677 - 0 . 5 7 3 0 . 181 CC 0 . 245 0 .274 0 .166 0 . 040 0 .572 CO - 0 . 8 2 5 - 0 . 492 - 0 . 6 23 - 0 . 5 8 3 - 0 . 0 9 1 CF - 0 . 8 0 9 - 0 . 5 50 - 0 . 632 - 0 . 5 1 0 - 0 . 2 87 CT - 0 . 6 6 8 - 0 . 423 - 0 . 5 02 - 0 . 4 1 1 - 0 . 1 2 5 OVERALL - 0 . 6 7 3 - 0 . 412 - 0 . 618 - 0 . 5 8 0 0 .337 RO CC CO CF CT RO 1 .000 CC - 0 . 2 3 7 1.000 CO 0 . 643 - 0 . 054 1.000 CF 0 . 584 - 0 . 229 0 .839 1 .000 CT 0 . 222 0 .127 0 .683 0 . 7 3 1 1.000 OVERALL 0 . 659 0 .345 0 .846 0 . 721 0 .706 OVERALL OVERALL 1 .000 139 Table 14. Correlation matrix from factor analysis of data of 12 days storage of Tr i a l 2 EXUDATE pH TSA TSN SPA EXUDATE 1. 000 pH -0. 562 1. 000 TSA 0. 277 0. .508 1.000 TSN 0. 346 0. .458 0.990 1 .000 SPA 0. 403 0. .362 0.942 0 .964 1 .000 SPN 0. 473 0. .267 0.909 0 .933 0 .969 TMAN 0. 184 0. .355 0.777 0 .739 0 .703 TVBN 0. 318 0. .209 0.504 0 .495 0 .475 WSP 0. 106 0, .604 0.811 0 .788 0 .763 SSP 0. 333 -0. .539 -0.042 -0 .008 0 .058 L 0. 400 -0. .523 0.070 0 .115 0 .170 a 0. 298 -0. .414 0.063 0 .083 0 .133 b 0. 283 -0. .120 0.247 0 .281 0 .311 OVERALL -0. 474 -0. .261 -0.864 -0 .880 -0 .880 RC -0. 197 -0. .387 -0.756 -0 .760 -0 .771 RO -0. 227 -0. .519 -0.905 -0. .898 -0 .868 CC -0. 620 0. .226 -0.351 -0 .401 -0 .440 CO -0. 454 -0. .228 -0.854 -0 .856 -0 .822 CF -0. 427 -0. .352 -0.878 -0 .897 -0 .912 CT -0. 586 0. .274 -0.372 -0 .403 -0 .419 SPN TMAN TVBN WSP SSP SPN 1. 000 TMAN 0. 712 1. 000 TVBN 0. 597 0. .452 1.000 WSP 0. 749 0, .599 0.516 1 .000 SSP 0. 151 0. .203 0.135 -0. .411 1 .000 L 0. 182 0. .161 -0.070 -0. .327 0 .735 a 0. 193 0. .275 -0.057 -0 .262 0 .709 b 0. 216 0. .003 -0.227 0 .071 0 .012 OVERALL -0. 853 -0. .705 -0.384 -0, .783 0 .043 RC -0. 647 -0. .504 -0.023 -0, .597 0 .164 RO -0. 840 -0. .811 -0.409 -0. .823 0 .096 CC -0. 413 -0. .172 -0.067 -0 .418 0 .122 CO -0. 828 -0. .762 -0.516 -0. .702 -0 .099 CF -0.893 -0. .629 -0.521 -0. .845 0 .099 CT -0. 509 -0. .525 -0.293 -0. .321 -0 .324 Continued next page Table 14 continued. L a ,L 1. ,000 a 0. ,746 1, .000 b 0. 387 0. .154 OVERALL -0. 081 0. .013 RC -0. 165 0. .052 RO 0. 061 -0. 024 CC -0. 115 0. .178 CO -0. 140 -0. 008 CF 0. 030 0. .110 CT -0. 224 -0, .377 RO CC RO 1. 000 CC 0. 394 1. .000 CO 0. 873 0, ,614 CF 0. 890 0. ,619 CT 0. 556 0. ,516 b OVERALL RC 1. .000 -0. 358 1.000 -0 .513 0.747 1 .000 -0, .220 0.915 0 .675 -0. 388 0.709 0 .476 -0. 282 0.950 0 .648 -0. 312 0.954 0 .711 -0. 057 0.613 0 .035 CO CF CT 1. .000 0. .904 1.000 0. .606 0.483 1 .000 141 Exudate volume also appeared to have some correlation with the cooked prawn meat texture score (r=-0.759 i n T r i a l 1 and r=-0.586 i n T r i a l 2, Table 12 and 14). Moisture content i n the muscle has been related to the juiciness of the meat. In this study, the CMAP prawns were found to have softer or mushier texture than the control and the NMAP prawns. A similar finding with f i s h f i l l e t s was also reported by Coyne (1933). 5 . 4 . Total psychrotrophic bacterial counts and total sulphide- producing psychrotrophic bacterial counts In this study, the CMAP extended the microbial lag phase to 4 days. I t was also very effective i n slowing the microbial growth rate during storage of the prawns and was much more effective than the NMAP. Since these bacteria appeared to be able to grow with or without oxygen, the majority of the microbial population present i n the raw material prawns must have been facultative anaerobes. However, i t was unlik e l y that these facultative anaerobes whould have been E. coli or Staphylococcus because these microorganisms are pathogens whose optimum growth temperature i s around 37°C. According to Gray et a l . , (1983), i t i s also unlikely that these pathogenic microorganisms which grow very slowly, i f at a l l , at re f r i g e r a t i o n temperature, w i l l grow under a carbon dioxide enriched-atmosphere even at abusive temperatures such as 10°C. According to Dainty et al., (1979), Pseudomonas spp., Aeromonas spp., and Enterobacteriaceae were capable of growing under low oxygen tension environment even though growth was not as vigorous as i n the nor-mal atmosphere as the storage continued. This probably was what happened i n the NMAP and the CMAP prawns. These bacteria including Pseudomonas 142 spp. and Alteromonas putrefaciens are dominant meat spoilage bacteria and also are sulphide producers (Lee et a l . , 1977). Since these sulphide producers played a very s i g n i f i c a n t role i n the spoilage of the prawns, their population under aerobic conditions has proved (by the stepwise discriminant analysis) to be an important indicator of quality stages of the prawns. Under the carbon dioxide and the nitrogen storage atmospheres, the psychrotrophic populations were markedly lower than that of the aerobic control treatment. I t i s clear that the difference i n the psychrotrophic population between the CMAP and the control treatments i s due to the bacteriostatic effect of carbon dioxide and the absence of oxygen. The same explanation may be used for the NMAP treatment where oxygen was absent and the carbon dioxide l e v e l i n the NMAP treatment increased during storage up to 13%. This small amount of carbon dioxide i n the NMAP bags l i k e l y inhibited psychrotrophic growth but not as strongly as i n the CMAP bags. Even though the concentration of carbon dioxide i n the NMAP treatment was high enough to i n h i b i t the growth of the psychrotrophs, i t was too low to induce a lag phase. 5.5. Trimethylamine-nitrogen concentration Dominant meat spoilage bacteria such as Pseudomonas spp. and Alteromonas putrefaciens are also TMAO reducers (Lee et al., 1977). The results showed that the NMAP system strongly favored the production of TMA. This i s i n agreement withobservations made by Watson (1939) that TMA was formed by anaerobic respiration of spoilage organisms. But the CMAP system did not favor the production of TMA even though i t i s considered an anaerobic environment. I n i t i t a l n h i b i t i o n of TMA 143 production i n the CMAP prawns was probably a result of the bacteriostatic effect of carbon dioxide. TMAN concentration i n the aerobic control prawns dropped during storage possibly because TMA was catabolized by some other microorganisms. Therefore, i n this study, under normal a i r atmosphere, TMA should be suitable as a measure of prawn quality for up to 2 weeks. 5.6. Total volatile basic nitrogen concentration The largest amount of TVBN was produced i n the control prawns f o l -lowed by the NMAP and the CMAP prawns respectively. This i s because the control treatment, which was a normal atmospheric environment i n addition to the low temperature of 1°C, strongly favored the growth of meat spoilage microorganisms. Microorganisms such as Pseudomonas species were shown to be capable of producing v o l a t i l e basic compounds (Cobb and Vanderzant, 1971). On the contrary, the CMAP system resulted i n the smallest production of v o l a t i l e basic compounds among a l l treatments i n this study. This was mainly the result of the inhibitory effects of carbon dioxide toward the normal meat-spoilage microorganisms. According to Yeh et al., (1978) and Satake et al., (1952), tissue enzymatic production of ammonia was evident over a wide pH range from s l i g h t l y acidic to a l k a l i n e . Yeh et al., (1978) also reported that there were s i g n i f i c a n t amounts of these enzymes (adenosine deaminase and AMP deaminase) i n shrimp muscle. Therefore, i t is speculated here that considerable amounts of v o l a t i l e basic compounds i n the CMAP prawns were the result of the a c t i v i t i e s of natural tissue enzymes i n prawns which were s t i l l active at s l i g h t l y a c i d i c pH. 144 Cobb and Vanderzant (1971) found that Pseudomonas species were strongly capable of producing v o l a t i l e basic compounds. The results i n this study showed that the best correlations of TVBN were to t o t a l anaerobic sulphide-producing psychrotrophic b a c t e r i a l count (r=0.597) and scores for flavour and for odour of cooked prawn meat (r=-0.521 and r=-0.516 respectively). This finding i s also i n general agreement with Gagnon and F e l l e r (1958). 5 . 7 . K-value and related compounds No ATP was detected i n prawn samples i n T r i a l 1. I t appears that ADP degradation was accelerated i n the CMAP prawns but was delayed i n the NMAP prawns. As a consequence, AMP i n the CMAP prawns was expected to be formed more rapidly than i n the other treatments. However, since there was no s i g n i f i c a n t difference i n AMP concentration i n prawns from a l l treatments of each sampling day, this may indicate that AMP was degraded more slowly i n the CMAP prawns than i n the NMAP prawns and was degraded most rapidly i n the control prawns. As a r e s u l t , IMP i n the NMAP prawns was formed faster than i n the CMAP prawns and thus was subjected to degradation sooner than that i n the CMAP prawns. IMP which was formed most rapidly i n the control prawns was degraded f i r s t . Inosine was formed accordingly to the IMP decomposition rate and hypoxanthine was also formed accordingly to the inosine decomposition rate. The results i n this study showed high c o r r e l a t i o n between K-value and IMP (r=-0.940). This supports the finding by Ehira (1976) that K-value was influenced most strongly by the IMP decomposition rate. In this study, the results showed that development of high K-values was faster i n the NMAP than i n the CMAP system. 145 5.8. Salt-soluble and WSP concentrations As a result of proteolysis, SSP were released from the muscle pro-teins and then were further hydrolyzed to form WSP. In this study, the results showed that proteolysis progressed most rapidly in the aerobic control prawns after approximately one week of storage when SSP started to decrease and WSP gradually increased and then peaked by day 12. In contrast, proteolysis in the CMAP and the NMAP prawns did not take place extensively until after about 2 weeks of storage since the concentrations of the SSP fractions in the CMAP and the NMAP prawns did not decrease until day 16. This is because growth of microorganisms, such as Pseudomonas species which are capable of degrading these proteins (Borton et al., 1970; Cobb and Vanderzant, 1971), were favored under the normal air atmosphere but were inhibited under carbon dioxide atmosphere. At the last period of storage of the aerobic control prawns, there was another increase in the SSP. This was possibly the result of the degradation of the major stromal proteins, collagen. Some Pseudomonas species were found to produce collagenases (Ockerman et al., 1969; Yada and Skura, 1981). The maximum concentrations of WSP fractions in the CMAP and NMAP prawns were much lower than that of the control prawns. The WSP fractions in the CMAP and the NMAP prawns increased at a much slower rate and peaked after the WSP fraction in the control prawns. The control prawns had the highest concentration of WSP; and the NMAP prawns had a slightly higher concentration of the WSP than the CMAP prawns. These results are in agreement with those reported by Borton et al., (1970) and Ockerman et al., (1969) that WSP increases with increase in tissue pH. 146 5.9. Colour As expected, the NMAP and the CMAP system prevented degradation of the colour (somewhat enhanced the redness and somewhat lightened the co-lour) of the raw, headless, shell-on pink prawns as indicated by the Hunter a and L values respectively. This was because the inert gas, nitrogen, of the NMAP system and the anaerobic conditions of both the NMAP and the CMAP systems provided protection from oxygen. Prawns under the nitrogen atmosphere had higher Hunter a values indicating that nitrogen gas provided better protection (for the carotenoid pigments against oxidation) than did the carbon dioxide i n the CMAP system. The Hunter L value was higher i n the CMAP prawns than i n the NMAP prawns. I t was probably because acidic conditions induced by the dissolved carbon dioxide causing some destruction of these carotenoid pigments i n the CMAP prawns. But for the NMAP prawns, there appears to be no explanation for this phenomenon. One p o s s i b i l i t y may be the carbon dioxide l e v e l i n the NMAP bags was s u f f i c i e n t to somehow destroy the carotenoid pigments but i t was too low to induce an acidic condition. Colour, which i s one of the major quality determinants for consumers of fresh prawns, when faded, resulted i n lowered color scores from the sensory panel. Intensity of yellowness i n colour of the NMAP prawns increased during the early stages of storage and la t e r decreased at the end of storage i n both t r i a l s . However, yellowness intensity of the CMAP prawn colour increased and reached higher values i n T r i a l 2 than i n T r i a l 1. Moreover, the results showed that changes i n the Hunter b value of a l l prawns i n both t r i a l s were dif f e r e n t . This maybe because the raw materi-147 a l prawns i n T r i a l 2 were more yellow i n colour to begin with or i t might be due to the stage of the prawns during th e i r spawning cycle. 5.10. Sensory characteristics The NMAP prawns had a very offensive putrid odour and a strong smell of hydrogen sulphide. Hydrogen sulphide production was probably the result of"amino acid decomposition. I t was reported that a high pH meat preserved i n vacuum, when spoiled, produced a strong putrid odour and hydrogen sulphide (Bern et a l . , 1976; Taylor and Shaw, 1977; Nicol et a l . , 1970). I t i s possible that TMA also made a s i g n i f i c a n t contribution to the putrid odour. The odour of the CMAP prawns was not as offensive as that of the NMAP prawns. Heating apparently influenced the odour score as the results showed that the scores for cooked prawn meat odour were s i g n i f i c a n t l y different i n both t r i a l s (P<0.001) from the scores for raw prawn meat odour. V o l a t i l e as well as non-volatile compounds i n prawns might be altered by heat i n the cooking process to produce strong odours. For instance, carotenoid, when subjected to heating, could result i n v o l a t i l e compounds such as toluene which has an unpleasant smell (Marty and Berset, 1986). The results showed that the scores for cooked prawn meat odour were generally lower than the scores for raw prawn meat odour. The score for cooked prawn meat flavour i n T r i a l 1 appeared to have a considerably good correlation with IMP (r=0.765) and hypoxanthine (r=-0.730). IMP may have influenced the cooked prawn meat flavour score by enhancing prawn flavour and hypoxanthine may influence the score for cooked prawn meat flavour by imparting the undesirable b i t t e r taste. 148 In both t r i a l s , the score for cooked prawn meat texture had considerably good correlation with exudate volume (r=-0.759 and r=-0.586 respectively, Tables 12 and 14) as mentioned e a r l i e r . The overall sensory score had i t s best correlation with the score for cooked prawn meat odour and flavour (r=0.846 and r=0.721 for T r i a l 1, r=0.950 and r=0.954 for T r i a l 2 respectively, Tables 12 and 14). Moreover, i t was also highly correlated to a l l microbiological variables i n T r i a l 2. This may imply that cooked prawn meat odour and flavour were the greatest contributors to the overall score and sensory quality of the prawns and were dependent on the microbiological quality of the prawns. 5.11. Shelf-life of the prawns In this study, the CMAP system for prawns increased the microbial lag phase to 4 days as well as s i g n i f i c a n t l y decreased psychrotrophic b a c t e r i a l growth rate during the logarithmic phase. The extension of s h e l f - l i f e of the prawns obtained by the CMAP system i s primarily a result of a f a i l u r e of the contaminating organisms, probably pseudomonads, to grow s u f f i c i e n t l y to cause spoilage. There was no increased lag phase for bacteria i n the NMAP prawns. However, microbial growth i n the NMAP prawns was retarded when compared to that i n the control prawns. A p r i n c i p a l factor i s the absence of oxygen and possibly the accumulation of carbon dioxide which has a marked i n h i b i t o r y effect on pseudomonads. S h e l f - l i f e of prawns i s dependent on the index used. For T r i a l 2, based on the t o t a l psychrotrophic bacterial count of lo g1 0 6.0 to 6.5, the s h e l f - l i v e s of the prawns i n this study were 1.5, 7, and 2 days for the control, the CMAP, and the NMAP prawns respectively. Based on the 149 TVBN concentration of up to 50 mg%, s h e l f - l i v e s of the prawns i n this study were 6 days for both the control and the NMAP prawns and 12 days for the CMAP prawns. These findings are generally i n agreement with Layrisse and Matches (1984). However, the s h e l f - l i f e of the pink prawns under carbon dioxide i n their study was longer. This i s because the raw material prawns i n this study were more heavily contaminated with microorganisms than ones used by Layrisse and Matches (1984). Moreover, the prawns i n this study were 4 days o ld when they arrived at the laboratory. I f a K-value of 20% i s used as a l i m i t for f i s h freshness (Ehira, 1976), the raw material prawns i n this study could not be considered fresh since the i n i t i a l K-value were 31.9%. Results from the factor analysis also showed that a l l microbiologi-c a l variables and TMAN concentration were the descriptors of Factor 1. Moreover, pH and SPA can be used to determine or predict s h e l f - l i f e or spoilage of the prawns (as the result from the stepwise discriminant analysis). Determination of pH i s simple, rapid, and inexpensive. I t was also s i g n i f i c a n t l y different among a l l treatments on every sampling day as well as during the whole storage. Moreover, i t was an important factor i n the effectiveness of carbon dioxide storage atmosphere i n i n h i b i t i n g microbial growth which resulted i n extended s h e l f - l i f e of the prawns. SPA presented direct information on the population of spoilage bacteria that contaminated the prawns. By putting i n the data for pH and SPA of the prawns, a plot of two canonical variables can be obtained. This plot helps as a geometric presentation of the quality of the prawns within the three regions of quality: good, acceptable, or unacceptable. 150 6. CONCLUSION 151 6. CONCLUSION In general, the results i n this study were i n agreement with Cobb et al., (1973) that the s h e l f - l i f e of the prawns was s i g n i f i c a n t l y l i m i t e d because of the a c t i v i t i e s of the microbial f l o r a ; and to a lesser extent because of the a c t i v i t i e s of the natural tissue enzymes of the prawns. Carbon dioxide was the only factor i n the packaging atmospheres that contributed the b a c t e r i c i d a l e f f e c t . While there was high concentration of carbon dioxide i n the CMAP bags, a small concentration of carbon dioxide did develop i n the NMAP bags as a result of microbial respiration and biochemical reactions. Any be n e f i c i a l effect of the MAP system was due to (1) exclusion of oxygen and (2) the concentration of carbon dioxide present i n the bag. Carbon dioxide, with i t s high s o l u b i l i t y i n the aqueous phase of the prawn muscle tissue was responsible for the si g n i f i c a n t pH drop. The reduction of the tissue pH caused a release of the sarcoplasm from the muscle. The CMAP prawns had the lowest tissue pH and the highest exudate volume. Effectiveness of these storage atmospheres i n i n h i b i t i n g microbial growth appears to relate to the amount of carbon dioxide gas i n the atmosphere. Facultative anaerobes appeared to be predominant i n the prawns i n this study since they could grow with or without the presence of oxygen. The growth of these microorganisms was markedly in h i b i t e d under the CMAP and NMAP system. These organisms were capable of producing sulphides, TMAO, v o l a t i l e bases, and had strong proteolytic a c t i v i t i e s , the development of TMAN, TVBN, SSP, and WSP occurred i n rel a t i v e to the growth of these psychrotrophic bacteria i n each treatment. An exception was that TMA production was favored under the 152 anaerobic conditions of the NMAP system. These results strongly indicated that these sulphide-producing psychrotrophic bacteria played a very significant role in spoilage of the prawns. Development of K-value was accelerated more by the NMAP than the CMAP as a consequence of the different decomposition rates of ADP as well as AMP in prawns in these two storage atmospheres. The development of K-value in the prawns was influenced most strongly by IMP decomposition rate. As indicated by the Hunter L and a values, colour of the prawns was well preserved under the NMAP and the CMAP systems due to their anaerobic conditions that protect the carotenoid pigments against oxidation by oxygen. While the acidic condition, induced by dissolved carbon dioxide, of the CMAP prawns caused destruction of the pigments, there appears to be no explanation for this phenomenon in the NMAP prawns except that the carbon dioxide level in the NMAP bags was sufficient to somehow destroy the carotenoid pigments in the prawn shell but i t was too low to induce an acidic condition in the tissue. These anaerobic conditions together with the presence of carbon dioxide resulted in the higher redness in the NMAP prawns than in the CMAP prawns and the lighter colour of the CMAP prawns compared to the NMAP prawns. Raw and cooked prawn meat odours were the main contributors to the sensory quality of the prawns. 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Chloramphenicol i n h i b i t s protein synthesis i n susceptible microorganisms by binding to the 50S ribosome subunits. As a r e s u l t , peptide formation i s i n h i b i t e d . Chloramphenicol i s active against various bacteria but inactive against fungi. A-2. Purpose The purpose of using chloramphenicol was to i n h i b i t growth of microorganisms i n the prawns hence eliminating the microbial a c t i v i t i e s which degrade the prawns. Therefore, only the degradation caused by prawn tissue enzymes could be examined. A-3. Concentration of the chloramphenicol solution Many experiments were carried out to determine the working concentration of the chloramphenicol solution and the treatment procedure. F i r s t , 50 gm headless shell-on prawns were soaked i n 500 ml of (1) deionized d i s t i l l e d water as the control, (2) 100 ppm chloramphenicol solution, and (3) 200 ppm chloramphenicol solution for 10 minutes at room temperature. After that the prawns were removed and the t o t a l plate counts were determined using trypticase soy ager and 21 °C incubation 165 temperature. The enumeration was done after 48 hours of incubation. The results were as follows: Treatments Control 100 ppm 200 ppm Total plate counts 6.10xl05 cfu/gm 1.14xl06 cfu/gm 8.60xl05 cfu/gm Second, 40 gm headless shell-on prawn were soaked i n 500 ml of (1) deionized d i s t i l l e d water as the control and (2) 100 ppm chloramphenicol solution for 30 minutes at room temperature. The solutions were discarded and each prawn sample was packed i n a s t e r i l e p l a s t i c bag and stored at 4°C for 24 hours. The t o t a l plate counts were determined. The results were as follows: Treatments Control 100 ppm Total plate counts 6.10xl05 cfu/gm 3.81xl05 cfu/gm Third, 50 gm headless shell-on prawns were soaked i n 500 ml of deionized d i s t i l l e d water as the control and (2) 100 ppm chloramphenicol solution for 24 hours at 4°C. The t o t a l plate counts were determined. The results were as follows: Treatments Control 100 ppm Total plate counts 6.10xl05 cfu/gm 4.46xl05 cfu/gm Fourth, 50 gm headless shell-on prawns were submerged i n 1000 ml of (1) deionized d i s t i l l e d water as the control, (2) 100 ppm chloramphenicol 166 solution, (3) 200 ppm chloramphenicol solution, and (4) 400 ppm chloramphenicol solution. Each was shaken under vacuum suction to allow the solutions to penetrate into the undershell areas and then l e f t soaking for 12 hours at 4°C. After soaking, the solutions were removed and each prawn sample was packed i n a s t e r i l e p l a s t i c bag, and stored for 4 days at 4°C. The t o t a l plate counts were determined. The results were as shown i n Table A - l . These results revealed that 200 ppm chloramphenicol solution with the treatment procedure as described i n the fourth experiment showed some i n h i b i t i o n as the microbial count decreased s l i g h t l y . When the chloramphenicol solution concentration increased to 400 ppm, the microbial count remained constant. This means that 400 ppm chloramphenicol solution solution e f f e c t i v e l y stopped the growth of the microorganisms present i n the prawn sample. Therefore, i t can be used to serve the purpose. A-4. Effect on the K-value of the prawns There was a concern as to whether this selected chloramphenicol solution treatment would affect the nucleotides within the prawn tissue i n any way. An experiment was set up to examine the effect of the choramphenicol treatment. Twenty-five grams of headless shell-on prawns were used per treatment (Table A - l ) . After the treatments, the muscle was blended and extracted for K-value determination as decribed i n section 3.5.5. Prior to i n j e c t i o n onto the HPLC column, 2 ml of the extract was mixed with 100 pil of the internal standard 5-bromouracil stock solution. The same d i l u t i o n process was carried out with the soaking solutions. Table A - l . Total aerobic psychrotrophic counts at 21°C 48 hours of fresh prawn samples subjected to control and chloramphenicol treatments Counts Zero = Counts after the soaking Counts Final = Counts after the whole treatment cfu/gm Counts Zero Counts Final Control Chloramphenicol 100 ppm Chloramphenicol 200 ppm Chloramphenicol 400 ppm 4.53xl03 2.20xl07 5.00xl02 3.41xl04 5.33xl03 4.20xl02 8.10xl02 8.lOxlO2 168 The K-values obtained from this experiment are shown i n Table A-2. It appeared that the i n i t i a l quality of the prawn used i n this experiment was poor and the chloramphenicol treatment sample had the lowest K-value among a l l the treated samples. From the r e s u l t s , i t appeared that the treatment procedure alone lowered the freshness as much as 27% K-value. However, this may be due to washing effect of the treatment. I t also appeared that chloramphenicol alone reduced the freshness as much as 16% K-value compared to the control treatment but "preserved" the freshness as much as 11% K-value compared to water treatment alone. K-values of both solutions appeared to be very s i m i l a r . I t was pos-s i b l e that the ATP and i t s related degraded compounds might have dis-solved into the solution i n i t i a l l y . The decision was then made that the chloramphenicol should not be used mainly because the whole treatment procedure alone decreased K-value by 27%. I t was not conclusive whether or how chloramphenicol affects the K-value. Furthermore, i t might cause overgrowth of non-susceptible organisms including fungi i n the samples. 169 Table A-2. The concentrations of ATP and i t s related compounds in prawn tissue extracts and soaking solutions Treatment 1 = Prawns without any treatments. Treatment 2 = 25 gm headless shell-on prawns were shaken for 2 minutes shaken under vacuum suction in 500 ml water and let soak for 12 hours at 4°C, then drained. Treated prawns were packed in a sterile plastic bag for 4 days at 4°C. Treatment 3 = 25 gm headless shell-on prawns were shaken for 2 minutes shaken under vacuum suction in 500 ml of 400 ppm chloramphenicol solution and soaked for 12 hours at 4°C, then drained. Treated prawns were packed in a sterile plastic bag for 4 days at 4°C. Treatment 4 = 25 gm headless shell-dn prawns were shaken for 2 minutes shaken under vacuum suction in 500 ml water, then drained. Prawns were packed in a sterile plastic bag for 4 days at 4°C. Solution 1 = The water drained out from treatment 2. Solution 2 = The chloramphenicol solution drained out from treatment 3. Millimoles/25gm HLSO prawn Extract IMP ATP ADP AMP Hx HxR % K value 1 19. .538 0, .000 2. .150 4. .292 48, .814 7, .942 68. .599 2 2. .347 0. .000 1. .485 0. ,500 89. .811 5. .280 95. ,643 3 7. .029 0, .000 1. .528 0. .000 39. .432 6, .203 84. .210 4 13. .967 0. .000 3. .145 0. ,911 122. .738 9. .973 88. .043 Soaking solution Millimoles/25gm HLSO prawn IMP ATP ADP AMP H x HxR % K value 1 24.776 0.000 5.468 0.000 615.897 0.000 95.319 2 30.822 0.000 6.762 0.000 517.910 14.631 93.408 APPENDIX B: SAMPLE MEANS AND STANDARD DEVIATIONS Table B-l. T r i a l 1: Sample means and standard deviations (n=6) of carbon dioxide, oxygen, and nitrogen concentrations (%) in the headspace atmospheres of CMAP bags on indicated sampling days DAY C02 02 N2 0 93, .11±1. 98 1. ,15±0. 59 5. .74±1. 47 3 81. ,01±5. 19 2. 22+0. .88 16. .77+4. 32 9 88, .09±2. ,69 0. .16±0. 04 11. .74+2. 66 18 88, .30+4. 56 0. ,11±0. 03 11. ,59±4. 53 Table B-2. Tri a l 1: Sample means and standard deviations (n=6) of carbon dioxide, oxygen, and nitrogen concentrations (%) in the headspace atmospheres of NMAP bags on indicated sampling days DAY C02 02 N2 0 0. .56±0. 15 1. 02±0. 11 98, .42±0. 15 3 2. .09+0. 25 0. ,70±0. .88 97. ,21±0. 71 9 3. 32±0. ,18 0. ,12+0. ,04 96. .56±0. 16 18 9. .04±0. 44 0. ,16±0. ,05 90. .80±0. 46 Table B-3. T r i a l 1: Sample means and standard deviations (n=15) of pHs of the control, the CMAP, and the NMAP prawns on indicated sampling days 173 DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 7.18±0.10 7.22±0.00 7.34±0.10 7.18±0.10 6.62±0.10 6.6110.00 6.65±0.00 6.7910.10 6.84+0.00 6.8910.10 7.1810.10 7.1010.10 7.1410.10 7.3510.10 7.3710.00 7.3310.10 7.3410.00 Table B-4. T r i a l 1: Sample means and standard deviations (n=3) of exudate volumes (ml) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 0.00±0.00 0.0010.00 0.0010.00 3 2.5710.70 7.4711.50 4.5710.70 6 3.3012.10 7.2011.80 5.1011.50 9 8.1712.50 3.5011.30 12 7.3311.50 6.4712.30 15 9.9311.80 7.1710.90 18 12.0011.50 5.2711.60 175 Table B-5. T r i a l 1: Sample means and standard deviations (n=6) of total aerobic psychrotrophic bacterial counts (Logi0 cfu/gm) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 5.71±0.10 5.71±0.10 5.71±0.10 3 7.1810.20 5.7410.10 6.7510.20 6 8.5910.10 6.1110.10 7.3210.10 9 6.6510.30 7.6710.10 12 7.0010.10 7.4310.00 15 7.0710.10 7.7810.30 18 7.2310.30 7.81+0.10 Table B-6. T r i a l 1: Sample means and standard deviations (n=6) of total anaerobic psychrotrophic bacterial counts (Log10 cfu/gm) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 5.80±0.10 5.80±0.10 5.80±0.10 3 7.1L+0.10 5.70±0.10 6.7L+0.10 6 8.4610.20 6.08±0.10 7.30+0.10 9 6.77±0.10 7.70±0.10 12 6.96±0.10 7.84+0.10 15 7.04+0.20 7.89±0.10 18 7.23±0.30 7.83±0.20 Table B-7. T r i a l 1: Sample means and standard deviations (n=6) of TMAN concentrations (gm/lOOgm tissue) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 0.50±0.03 0.5010.03 0.50±0.03 3 0.9010.51 0.4110.18 2.6212.77 6 6.9911.52 4.4814.29 9.0712.57 9 5.48+4.82 30.3716.05 12 21.4415.56 40.60+3.58 15 26.2018.13 58.2015.44 18 41.6910.62 65.0112.63 178 Table B-8. T r i a l 1: Sample means and standard deviations (n=6) of ADP concentrations (nanomoles/ml extract) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 7392.17±2569.76 7392.17±2569.76 7392.1712569.76 3 4717.051 471.86 4028.491 71.48 6470.581 936.84 6 4041.4311000.39 3486.981 811.70 5621.2211453.77 9 2780.831 966.31 4006.561 639.53 12 3284.111 721.70 824.5111409.06 15 3005.1211014.48 2218.4811619.36 18 2792.561 660.12 1899.461 270.66 179 Table B-9. T r i a l 1: Sample means and standard deviations (n=6) of AMP concentrations (nanomoles/ml extract) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 37303.31±5661.37 37303.31±5661.37 37303.31±5661.37 3 18093.41±1608.86 23119.19±6344.96 22772.34±6056.19 6 5614.33± 662.49 7789.81± 111.27 8162.80±1764.03 9 5668.90±1504.82 4003.93± 700.42 12 2910.35± 433.05 3249.40±1562.68 15 2057.23±1408.41 1679.44± 789.79 18 1747.69± 665.70 1368.69± 492.82 180 Table B-10. T r i a l 1: Sample means and standard deviations (n=6) of IMP concentrations (nanomoles/ml extract) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 51516.30±13281.76 51516.30±13281.76 51516.30±13281.76 3 35131.96± 3832.63 39742.30±11934.51 43987.30± 265.87 6 15671.61± 7949.21 27810.78± 5994.80 21736.31± 685.88 9 18957.11+ 4697.93 2990.83± 2632.64 12 15824.26+ 4209.94 7826.04± 6801.99 15 9516.56± 7286.93 0.00± 0.00 18 6535.33± 5764.07 0.00+ 0.00 181 Table B - l l . T r i a l 1: Sample means and standard deviations (n=6) of inosine concentrations (nanomoles/ml extract) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 37548.35±9743.14 37548.35± 9743.14 37548.35±9743.14 3 34763.81±2843.56 38614.07± 4654.66 44297.14±8374.08 6 27126.19+ 816.16 41444.89±11352.35 37769.53±3214.53 9 44311.13± 3397.51 14768.67±4609.46 12 31738.83±21514.73 12162.87+7014.71 15 26189.95+14771.40 3097.89±3593.48 18 20212.61± 6177.85 1896.95±3285.61 182 Table B-12. T r i a l 1: Sample means and standard deviations (n=6) of hypoxanthine concentrations (nanomoles/ml extract) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 8086.95±3239.63 8086.95± 3239.63 8086.95± 3239.63 3 13115.36±7349.20 18991.42± 5115.77 11563.57± 5633.67 6 35991.61+9966.30 43859.93±19196.37 45284.63± 9467.29 9 52262.38124321.56 101734.42±32913.38 12 76221.611 6610.78 103246.361 8057.00 15 75252.02120470.06 108282.471 5248.81 18 85519.191 3367.70 105050.68110211.14 183 Table B-13. Tr i a l 1: Sample means and standard deviations (n=6) of K-values (%) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 31.92±1.81 31.92±1.81 31.92±1.81 3 45.01±6.28 46.62±2.65 42.94±2.74 6 71.66±9.02 68.36±3.86 68.37±3.25 9 77.30±7.16 91.28±3.66 12 83.03±1.86 89.08±6.61 15 88.44±6.44 96.68±1.92 18 90.98±5.13 97.03±0.36 184 Table B-14. Tr i a l 1: Sample means and standard deviations (n=30) of Hunter L values of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 37.77±0.53 37.77±0.53 37.7710.53 3 37.82±0.38 39.6110.65 39.5410.65 6 37.74+0.80 40.5311.43 40.0110.14 9 40.55+0.34 40.1510.82 12 41.20+0.24 40.2611.02 15 41.29+0.95 41.15+0.67 18 41.5710.52 41.4510.68 185 Table B-15. T r i a l 1: Sample means and standard deviations (n=30) of Hunter a values of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 9.4710.37 9.47±0.37 9.4710.37 3 9.8010.58 10.7611.99 11.9310.47 6 9.3010.35 11.9711.27 13.1410.25 9 11.6210.22 15.5910.56 12 12.46+0.54 15.2810.80 15 12.8310.63 15.4011.09 18 12.65+0.54 15.5011.19 Table B-16. T r i a l 1: Sample means and standard deviations (n=30) of Hunter b values of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 10.27±0.36 10.2710.36 10.2710.36 3 11.08+0.17 10.9110.48 11.3910.52 6 11.2010.23 11.4010.49 11.7110.32 9 10.6410.18 12.1010.12 12 11.5010.01 11.8510.09 15 11.4810.11 11.7610.45 18 11.8710.20 11.6410.54 187 Table B-17. T r i a l 1: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for colour of raw prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 5.86±1.88 7.00±1.41 6.00+1.93 5.86±1.88 6.14±2.03 5.14±2.10 6.43±1.29 5.43±1.76 7.14±1.12 5.00±1.77 5.86±1.88 5.14±1.96 5.29±1.58 6.00±2.20 6.86±0.64 7.29±0.88 6.57±1.29 Table B-18. T r i a l 1: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for odour of raw prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 7.14±1.64 7.2911.48 5.8611.64 7.14±1.64 7.1411.64 6.29+1.67 6.8611.46 6.2911.48 7.2911.39 5.5711.50 7.14+1.64 6.4312.13 5.5711.59 4.5711.76 5.7111.91 6.2911.67 4.7112.60 Table B-19. Tri a l 1: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for colour of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 5.86±2.53 6.14±2.17 6.00+1.93 5.86±2.53 4.00±1.77 5.57+2.13 5.86+2.10 4.29±2.25 5.71±1.91 4.14±2.36 5.8612.53 5.8611.96 6.00+2.14 5.4312.32 7.8610.83 7.4310.49 7.5710.90 190 Table B-20. T r i a l 1: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for odour of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 6.71±1.48 5.86±1.96 4.71+1.91 6.7111.48 4.8611.46 4.1411.81 4.5711.99 4.14+2.59 4.2912.05 4.4311.76 6.7111.48 6.8611.96 3.5711.76 4.0012.20 4.00+2.27 5.1412.29 3.7111.39 Table B-21. T r i a l 1: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for flavour of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 6.86±0.64 5.71±1.16 5.86±1.81 6.86±0.64 6.43±1.68 6.00±1.07 5.00±2.14 4.57±1.76 5.14±2.36 5.57±1.40 6.86±0.64 6.71±1.75 5.4311.92 5.14±1.96 4.86±2.23 6.14±2.10 4.29±1.58 192 Table B-22. Tr i a l 1: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for texture of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 7.4310.73 7.2910.88 7.1410.64 7.4310.73 6.7111.67 6.7110.70 5.4312.32 6.2911.58 5.8612.17 5.8611.25 7.43+0.73 7.4311.50 6.7111.58 7.0011.31 6.7111.67 7.0011.41 6.0011.69 193 Table B-23. Tri a l 1: Sample means and standard deviations (3 bags/sample and 7 judges) of overall sensory scores of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3 6 9 12 15 18 6.64±1.00 6.55±0.95 5.93+1.04 6.6411.00 5.8811.22 5.6410.91 5.6911.13 5.1711.05 5.9011.08 5.1010.96 6.64+1.00 6.4011.39 5.4311.11 5.3611.04 6.0011.02 6.5510.72 5.4811.05 Table B-24. T r i a l 2: Sample means and standard deviations (n=6) of carbon dioxide, oxygen, and nitrogen concentrations (%) in the headspace atmospheres of CMAP bags on indicated sampling days DAY C02 02 N2 0 94.57±1.84 1.04±0.36 4.39±1.48 8 84.91±5.47 0.46±0.51 14.63±5.43 16 83.83±1.96 0.30±0.02 15.87±1.95 28 82.20±1.34 0.40±0.08 17.41±1.36 Table B-25. T r i a l 2: Sample means and standard deviations (n=6) of carbon dioxide, oxygen, and nitrogen concentrations (%) in the headspace atmosphere of NMAP bags on indicated sampling days DAY C02 02 N2 0 0. ,95±0. 06 1. ,63±0. 60 97. .42±0. 65 8 8. .86+7. 23 0. .10+0. 02 91. .04+7. 22 16 6. .67±0. 33 0. .12±0. 04 93. .21+0. 36 28 12. .95±3. 93 0. .51+0. 50 86, .54±4. 42 Table B-26. T r i a l 2: Sample means and standard deviations (n=15) of pHs of the control, the CMAP, and the NMAP prawns on indicated sampling days Day CONTROL CMAP NMAP 0 7.4510.14 7.4510.14 7.4510.14 4 7.2910.05 6.45+0.05 7.10+0.04 8 7.7610.12 6.5710.07 7.36+0.05 12 8.0910.04 6.8310.27 7.4810.03 16 8.29+0.08 6.8410.05 7.53+0.11 20 8.0010.51 6.8210.35 7.2710.03 24 8.4010.05 6.7110.00 7.3510.12 28 8.4210.04 6.8210.05 7.3410.01 Table B-27. T r i a l 2: Sample means and standard deviations (n=3) of exudate volumes (ml) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 0. .00±0. 00 0. .00±0, .00 0. .00±0. 00 4 6. ,37±1. .42 8. .17±2. 47 6. .90±1. .47 8 4. ,27±0. 60 9. .93+1. 72 3. ,97±2. 15 12 5. 43+0. 47 7. ,77±5. 35 4. .80±1. .18 16 3. 93+0. 83 5. .87±1. .70 5. .97±3. 56 20 5. .73±1. .85 6. 03+2. 99 8. ,17±1. .04 24 11. .30±1. .39 10. ,27±2. 27 7. ,03±1. .76 28 13. ,90±3. 18 10. ,60±6. 16 6. .5311. .77 198 Table B-28. T r i a l 2: Sample means and standard deviations (n=6) of total aerobic psychrotrophic bacterial counts (Log10 cfu/gm) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 5, ,88±0. 57 5. 8810. .57 5. .88±0. 57 4 7. ,63±0. 21 5. .84±0. 41 7. ,44±0. 24 8 8, .51±0. 06 6, ,61±0. 54 7. .90±0. 13 12 9. .95±0. 12 8. .10±0. 25 9. ,23±0. 08 16 9. .35±0. 08 7. .42±0. 59 7. ,97±0. 10 20 10. .00+0. 00 8. .94±0. 86 11. ,65±0. 00 24 28 9. ,29±0. 04 8. .08±0. 06 8. .18±0. 06 199 Table B-29. T r i a l 2: Sample means and standard deviations (n=6) of total anaerobic psychrotrophic bacterial counts (Logi0 cfu/gm) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 5. ,69±0. 61 5. ,69±0, .61 5. .69±0. 61 4 7. ,71±0. 23 5. .89±0, .40 7, ,50±0. 25 8 8. .5910. .05 6. ,85±0. 58 7, ,97±0. 09 12 9. ,89±0. 06 8. .14±0. 19 8. ,91±0. 05 16 9. .34±0. 07 7. .60±0. 67 7. .92±0. 12 20 9. ,65±0. 26 24 28 9. ,33±0. ,01 8. ,14±0. 10 8. .23+0. ,06 200 Table B-30. T r i a l 2: Sample means and standard deviations (n=6) of total aerobic sulphide-producing psychrotrophic bacterial counts (Log^ cfu/gm) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 1. ,78±0. 35 1. .78±0. 35 1. ,78±0. ,35 4 4. 23±0. ,03 2. .43±0. 49 4. ,20±0. ,07 8 4. .98±0. 11 3. ,31±0. 03 5. .1110. .02 12 6. ,77±0. 19 5. ,26±0. 12 4. ,99±0. 14 16 4. ,85±0. 21 4. .60±0. 63 5. .11±0. ,12 20 6. .19+1, .12 24 28 5. .41±0. 00 5. ,29±0. 09 ±0. 06 Table B-31. T r i a l 2: Sample means and standard deviations (n=6) of total anaerobic sulphide-producing psychrotrophic bacterial counts (Log10 cfu/gm) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 0. .76±0. 32 0. ,76±0. ,32 0. ,76±0. 32 4 4. .12±0. 08 2. ,38±0. ,87 4. ,27±0. ,20 8 4, ,09±0. 09 2. 68±0. 50 4. .91±0. 22 12 6. .58±0. 32 4. ,63±0. ,08 5. .22±0. 21 16 5. ,12±0. 17 4. ,30±0. ,76 4. ,92±0. 13 20 6. ,19±1. 12 24 28 4, .40±0. 00 5. 30±0. 09 5. ,22±0. 13 Table B-32. T r i a l 2: Sample means and standard deviations (n=6) of TMAN concentrations (gm/lOOgm tissue) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 0. .06+ 0. .01 0. ,06± 0. .01 0. ,06± 0. .01 4 2. . 50± 0. .78 2. .12+ 0. .86 3. ,48± 0. .70 8 12. ,50± 0. .95 3. .68+ 2. .00 24. ,34± 9. .45 12 27, .02± 2. .84 15. .26+ 9, .78 43. .70± 8. .15 16 33, .94± 1. 15 31. ,66± 6, .02 48. ,56± 7, .88 20 22, ,20± 8. .00 37. ,46±10. .20 58. ,04± 6. .80 24 35, .70± 2. .08 55. ,80± 3. .25 74. ,00±15. 00 28 46, .40±14. .66 64, .00111, .15 86. .94±24 .07 Table B-33. T r i a l 2: Sample means and standard deviations (n=6) of TVBN concentrations (gm/lOOgm tissue) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 8. .65±0. 12 8. ,65±0. 12 8. .65±0. 12 4 21. .67±0. 15 16. .94±0. 11 18. ,90±0. ,06 8 62. .44+0. 20 31. .92±0. 14 64. .82+0. ,14 12 95. ,06±0, .10 47. ,46±0. 15 103, ,60±0. 21 16 155, .40±0. 09 98. .70±0. 30 116, ,20±0. ,16 20 206. ,92±0. 65 106. ,96±0. 98 181, .02±0. 68 24 226. .38±0. 47 104. .16±0. 45 128. .80±0. 32 28 227, .92±0, .56 109, ,06±0. 16 116, .06±0. 40 204 Table B-34. T r i a l 2: Sample means and standard deviations (n=6) of water-soluble protein concentrations (gm/lOOgm tissue) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 . 2.77±0.21 2.77±0.21 2.77±0.21 4 3.8210.15 3.15±0.23 4.39±0.39 8 4.4L+0.15 2.85±0.21 3.9L+0.34 12 10.39±0.20 3.94±0.59 5.56±0.44 16 9.83+2.21 7.39±0.67 7.96±0.59 20 24 4.48+0.53 4.05+0.20 6.41±0.95 28 3.52±1.03 3.56±1.57 2.46±0.35 205 Table B-35. T r i a l 2: Sample means and standard deviations (n=6) of salt-soluble protein concentrations (gm/lOOgm tissue) of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 3. 92+0. 49 3. 9210. 49 3. 8210. 49 4 4. .49+0, .29 5. .46±0. .13 5. 1310. 40 8 4. .37+0. 33 5. ,55±0, .32 5. 6910. 55 12 2. ,59±0. .44 5. 4810. 45 5. 6910. 55 16 2. .84±0. .72 5. 8810. 20 5. .7110. 72 20 24 5. .86±0. .83 5. 1210. 14 4. .1910. 94 28 0. .05+2. 44 2. .6510. 50 2. .6911. 07 206 Table B-36. Tr i a l 2: Sample means and standard deviations (n=30) of Hunter L values of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 37.4110.43 37.41±0.43 37.4110.43 4 38.89+2.06 38.6111.76 38.8910.86 8 38.1210.15 40.1910.86 39.8110.40 12 35.7910.26 42.0611.39 39.8910.42 207 Table B-37. T r i a l 2: Sample means and standard deviations (n=30) of Hunter a values of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 12.07±0.43 12.0710.43 12.07+0.43 4 11.6611.42 12.6310.62 13.9410.42 8 11.46+0.48 13.83+1.43 15.3010.94 12 10.8410.71 14.0910.34 14.1610.21 Table B-38. T r i a l 2: Sample means and standard deviations (n=30) of Hunter b values of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 12.03±0.30 12.03±0.30 12.03±0.30 4 12.07±0.78 12.09±0.51 12.41±0.88 8 13.16±0.33 12.8610.41 12.11±0.34 12 12.42±0.53 13.1510.74 12.0510.48 T a b l e B -39 . T r i a l 2: Sample means and s t a n d a r d d e v i a t i o n s (3 b a g s / s a m p l e and 7 j u d g e s ) o f s c o r e s f o r c o l o u r o f raw prawn meat o f t he c o n t r o l , t h e CMAP, and t he NMAP prawns on i n d i c a t e d s a m p l i n g days DAY CONTROL CMAP NMAP 0 6 .33±1 .60 6 .33±1.60 6 .33±1 .60 4 6 .17±2 .34 7 . 1710 . 90 6 . 0 011 . 00 8 4 . 5 0 1 1 . 6 1 5 . 8311 .34 6 . 0 011 . 29 12 4 . 1 7 1 1 . 6 7 3 . 8310 . 90 5 . 4 011 . 50 Table B-40. T r i a l 2: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for odour of raw prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 7.33±1.25 7.33±1.25 7.33±1.25 4 6.67±1.80 7.00±0.58 6.00±1.63 8 4.00±1.91 5.83±1.77 4.67±0.75 12 2.33±1.11 6.00±1.73 3.50±1.71 211 Table B-41. T r i a l 2: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for colour of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 6. 67+1. 80 6. .67±1. .80 6. 67+1. 80 4 5. .67±1. .89 4. .50±1. .50 6. 83+1. 86 8 5. ,33±2. 05 4. .17±2. 11 6. .67±0. 75 12 3. ,83±2. 19 4. ,50±3. 55 - 6. ,00±1. 41 Table B-42. Trial 2: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for odour of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 5.33±1.49 5.33±1.49 5.33±1.49 4 4.0012.38 3.8312.03 4.5012.36 8 2.8311.34 4.0011.67 4.1712.27 12 2.3311.25 3.5012.29 2.1711.34 Table B-43. Tr i a l 2: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for flavour of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 6.67±0.94 6.67±0.94 6.6710.94 4 5.1712.61 5.1711.95 5.1711.67 8 3.8312.03 5.6011.20 5.0011.79 12 2.5011.38 4.6712.21 4.5011.50 214 Table B-44. T r i a l 2: Sample means and standard deviations (3 bags/sample and 7 judges) of scores for texture of cooked prawn meat of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 6.67±1.25 6.67±1.25 6.67±1.25 4 6.33±1.80 5.17±2.11 6.00±1.41 8 6.20±1.72 4.80±1.47 5.33±1.97 12 5.3311.80 6.33±1.60 5.0012.08 215 Table B-45. T r i a l 2: Sample means and standard deviations (3 bags/sample and 7 judges) of overall sensory scores of the control, the CMAP, and the NMAP prawns on indicated sampling days DAY CONTROL CMAP NMAP 0 6.5010.92 6.5010.92 6.5010.92 4 5.6711.83 5.4711.30 5.7511.07 8 4.4511.06 5.0411.10 5.3110.43 12 3.4211.28 4.8111.52 4.4310.97 

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